Composition comprising xanthoceras sorbifolia extracts, compounds isolated from same, methods for preparing same and uses thereof

ABSTRACT

This invention provides compositions, methods and process of producing extracts from  Xanthoceras sorbifolia . The extract comprises alkaloids, coumarins, saccharides, proteins, polysaccharides, glycosides, saponins, tannins, acid, flavonoids and others. The composition can be used for treating cancer, arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud&#39;s syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder, impotence and premature ejaculation; for preventing cerebral aging; for improving memory, cerebral functions; or for curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer&#39;s disease, autism, brain trauma, Parkinson&#39;s disease or other diseases caused by cerebral dysfunctions, and treating arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud&#39;s syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder. This invention provides compounds comprising at least one sugar, a triterpene, such as Sapogenin, and at least one side chains at Carbon 21 and 22, such as Angeloyl groups. The compounds of the present have various pharmaceutical and therapeutic applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/718,575, Filed Dec. 18, 2012, which is a continuation of U.S. Ser. No. 12/392,795, Filed Feb. 25, 2009, U.S. Pat. No. 8,334,269, issued Dec. 18, 2002, which is a continuation of U.S. Ser. No. 10/906,303, Filed Feb. 14, 2005, U.S. Pat. No. 7,524,824, issued Apr. 28, 2009, which is a Continuation-In-Part application of International Application No. PCT/US04/43465, Filed Dec. 23, 2004, which is a Continuation-In-Part application of PCT/US04/33359, Filed Oct. 8, 2004, which claims benefit of U.S. Ser. Nos. 60/617,379, Filed Oct. 8, 2004, 60/613,811, Filed Sep. 27, 2004, 60/607,858, Filed Sep. 7, 2004, 60/532,101, Filed Dec. 23, 2003, and 60/509,851, Filed Oct. 9, 2003. Furthermore, U.S. Ser. No. 10/906,303, Filed Feb. 14, 2005, is a Continuation-In-Part of U.S. Ser. No. 10/471,384, Filed Sep. 4, 2003, U.S. Pat. No. 7,189,420, issued Mar. 13, 2007, which is a National Stage of Intl App'l No. PCT/IB02/04750, Filed Aug. 28, 2002, which is a continuation-in-part of U.S. Ser. No. 09/944,805, Filed Aug. 31, 2001, U.S. Pat. No. 6,616,943, issued Sep. 9, 2003. The entire contents of these preceding applications are hereby incorporated by reference into this application.

Throughout this application, various publications are referenced. Disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

This invention relates to extracts from a plant called Wenguanguo (Xanthoceras Sorbifolia Bunge), their usages and functions, and methods of their preparation.

BACKGROUND OF THE INVENTION

Wenguanguo is a species of the sapindaceae family. Its scientific name is Xanthoceras sorbifolia Bunge. Wenguanguo is the common Chinese name; others are Wenguanngguo, Wenguanmu, Wenguanhua, Xilacedeng and xilasendeng. Goldenhorn and Yellowhorn are its common English names. Wenguanguo is grown in Liaoning, Jilin, Hebei, Shandong, Jiangsu, Henan, Shanxi, Shaanxi, Gansu, Ningxia and Inner Mongolia, China. Its seeds, leaves and flowers are edible and its seeds have been used as a folk medicine to treat enuresis for centuries. Its branches and woods are also used as a folk medicine.

Chinese patent applications CN 1092991A and CN 1092992A discussed the methods for producing a medicine from Wenguanguo kernel powder for curing enuresis and enhancing cerebral functions. Chinese patent CN 1052636C discussed a method for producing a medicine with ethanol extract from the Wenguanguo kernel powder for curing enuresis and enhancing cerebral functions. Journal of Shenyang University of Pharmacy (2001), 18(1), 53-56 disclosed the n-butanol extract from the wood of Wenguanguo, which has anti-inflammatory effect.

United States Patent Application Publication No. 20030096030 discussed the extracts from the husks of Wenguanguo which are Bunkankasaponin A. B. C. D and two sterols for preventing cerebral aging, improving cerebral functions and curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence, and increasing the body's ability to resist the activity of glycosuria.

United States Patent Application Publication No. 20030082293 disclosed the extracts Bunkankasaponin A. B. C. D, crude fats, crude protein and sugars from the shell of Wenguanguo.

U.S. Pat. No. 6,616,943, issued on Sep. 9, 2003, discussed the composition comprising Wenguanguo combined extracts and the methods for preparing them and uses thereof. The methods for preparing the combined extract from the husks comprise the following steps: extracting Wenguanguo husks with an organic solvent (e.g. ethanol) to form an organic (e.g. ethanol) extract; removing the organic solvent (e.g. ethanol) from the organic (e.g. ethanol) extract to form aqueous extracts; and drying and sterilizing the aqueous extracts to form the combined extracts. The combined extracts contain saponins, saccharides, proteins and others. The extracts can be used for producing medicines or health foods for preventing cerebral aging, improving memory, improving cerebral functions and curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer's disease, autism, brain trauma, Parkinson's disease and other diseases caused by cerebral dysfunction. The medicines or health foods further comprise Vitamin B, Vitamin D, K, anti-oxidant, Cordyceps or its extracts, gingko or its extracts, Echinacea or its extracts, Huperzine A, folic acid, amino acids, creatine, fiber supplement or a combination thereof.

Yingjie Chen, Tadahiro Takeda and Yukio Ogihara in Chem. Pharm. Bull 33(4)1387-1394(1985) described a study on the constituent of Xanthoceras sorbifolia Bunge. See Section V. Saponins from the Fruits of Xanthoceras sorbifolia. Four new saponins were isolated from the fruits of Xanthoceras sorbifolia Bunge. The structures of these saponins are bunkankasaponins A, B, C and D:

-   (1)     22-O-acetyl-21-O-(4-O-acetyl-3-O-angeloyl)-β-D-fucopyranosyl-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]protoaecigenin -   (2)     22-O-acetyl-21-O-(3,4-di-O-angeloyl)-β-D-fucopyranosyl-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]protoaecigenin -   (3)     28-O-acetyl-21-O-(4-O-acetyl-3-O-angeloyl)-β-D-fucopyranosyl-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]protoaecigenin -   (4)     28-O-acetyl-21-O-(3,4-di-O-angeloyl)-β-D-fucopyranosyl-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]protoaecigenin

Yingjie Chen, Tadahiro Takeda and Yukio Ogihara in Chem. Pharm. Bull 33(3)1043-1048(1985) described studies on the constituent of Xanthoceras sorbifolia Bunge. See Section IV. Structures of the Miner Prosapogenin. The prosapogenins from the partial hydrilyzate of fruit saponin of Xanthoceras sorbifolia were examinated, and are characterized as:

-   16-O-acetyl-21-O-(3,4-di-O-angeloyl-β-D-fucopyranosyl)     protoaecigenin -   22-O-acetyl-21-O-(3,4-di-O-angeloyl-β-D-fucopyranosyl)     protoaecigenin 3-O-β-D-glucuronopyranoside

Yingjie Chen, Tadahiro Takeda and Yukio Ogihara in Chem. Pharm. Bull 33(1)127-134(1985) describe studies on the constituent of Xanthoceras sorbifolia Bunge. See Section III. Minor Prosapogenins aponins from the Fruits of Xanthoceras sorbifolia Bunge. The structure of 3 minor prosapogenins, obtained by acid hydrolysis of the crude saponin faction, were characterized as:

-   21-O-(3,4-di-O-angeloyl)-β-D-fucopyranosyltheasapogenol B -   21-O-(4-O-acetyl-3-O-angeloyl)-β-D-fucopyranosyltheasapogenol B -   21-O-(4-O-acetyl-3-O-angeloyl)-β-D-fucopyranosyl-22-O-acetylprotoaescigenin

Yingjie Chen, Tadahiro Takeda and Yukio Ogihara in Chem. Pharm. Bull 33(4)1387-1394(1985) described a study on the constituent of Xanthoceras sorbifolia Bunge. See Section II. Major Sapogenol and prosapogenin from the Fruits of Xanthoceras sorbifolia.

Laurence Voutquenne, Cecile Kokougan. Catherine Lavaud, Isabelle Pouny, Marc Litaudon. Triterpenoid saponins and Acylated prosapogenins from Harpullia austro-caledonica. Phytochemistry 59 (2002) 825-832

Zhong Jaing, Jean-francois Gallard, Marie-Therese Adeline, Vincent Dumontet, Mai Van Tri, Thierry Sevenet, and Mary Pais Six Triterpennoid Saponins from Maesa laxiflora. J. Nat. Prod. 1999, 62, 873-876

Young Seo, John M. Berger, Jennine Hoch, Kim M Neddermann, Isia Bursuker, Steven W. Mamber and David G. Kingston. A new Triterpene Saponin from Pittosporum viridiflorum from the Madagascar Rainforest. J. Nat. Prod. 2002, 65, 65-68

Xiu-Wei Yang, Jing Zhao, Xue-Hui Lui, Chao-Mei Ma, Masao Hattori, and Li He Zhang Anti-HIV-1 Protease Triterpenoid Saponins from the Seeds of Aesculus chinensis. J. Nat. Prod. 1999 62, 1510-1513

Yi Lu, Tatsuya Umeda, Akihito Yagi, Kanzo Sakata, Tirthankar Chaudhuri, D. K. Ganguly, Secion Sarma. Triterpenoid Saponins from the roots of the tea plant (Camellia sinensis var. Assamica). Phytochchemistry 53 (2000) 941-946

Sandra Apers, Tess E. De Bruyne, Magda Claeys, Arnold J. Viletinck, Luc A. C. Pieters. New acylated triterpenoid saponins from Maesa laceceolata. Phytochemistry 52 (1999) 1121-1131

Ilaria D'Acquarica, Maria Cristina, Di Giovanni, Francesco Gasparrini, Domenico Misiti, Claudio D'Arrigo, Nicolina Fagnano, Decimo Guarnieri, Giovanni Iacono, Giuseppe Bifulco and Raffaele Riccio. Isolation and structure elucidation of four new triterpenoid estersaponins from fruits of the Pittosporumtobira AIT.

Tetrahedron 58 (2002) 10127-10136

Cancer cells are defined by two heritable properties: (1) they reproduce in defiance of normal restraints on cell division; and (2) they invade and colonize territories normally reserved for other cells.

Cancers require mutations of many genes to develop, and they are classified according to the tissue and cell type from which they arise. Cancers arising from epithelial cells are named carcinomas; those arising from connective tissue or muscle cells are named sarcomas. In addition, there are cancers called leukemias, which are derived from hemopaietic cells; and cancers derived from cells of the nervous system.

Cancers originating from different types of cells are, in general, very different diseases. Each cancer has characteristics that reflect its origin. Even when a cancer has metastasized and proliferated out of control, its origins can be traced back to a single, primary tumor. Therefore it is important to develop drugs against target cells with a specified character.

Ovarian cancer is the 5th leading cause of cancer death in women and the leading cause of death from gynecologic. In the United States, females have a 1.4 to 2.5% (1 out of 40-60 women) lifelong chance of developing ovarian cancer. Older women are at highest risk. More than half of the deaths from ovarian cancer occur in women between 55 and 74 years of age and approximately one quarter of ovarian cancer deaths occur in women between 35 and 54 years of age.

Ovarian cancer is disproportionately deadly for a number of reasons. First, symptoms are vague and non-specific, so women and their physicians frequently attribute them to more common conditions. By the time the cancer is diagnosed, the tumor has often spread beyond the ovaries. Also, ovarian cancers shed malignant cells that frequently implant on the uterus, bladder, bowel, and lining of the bowel wall (omentum). These cells can begin forming new tumor growths before cancer is even suspected. Second, because no cost-effective screening test for ovarian cancer exists, more than 50 percent of women with ovarian cancer are diagnosed in the advanced stages of the disease.

This invention provides compounds or compositions extracted from Xanthoceras Sorbifolia or synthesized which have substantial potency against ovarian cancer.

SUMMARY OF THE INVENTION

In accordance with these and other objects of the invention, a brief summary of the present invention is presented. Some simplifications and omission may be made in the following summary, which is intended to highlight and introduce some aspects of the present invention, but not to limit its scope. Detailed descriptions of a preferred exemplary embodiment adequate to allow those of ordinary skill in the art to make and use the invention concepts will follow in later sections.

The invention provides a compound comprising the following structure, with the formula of C₅₇H₈₈O₂₃ and the name of 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β, 15α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene, also known as Xanifolia-Y This compound was isolated from Xanthoceras sorbifolia.

This invention provides a compound comprising the following structure, with the formula of C₆₅H₁₀₀O₂₇ and the name of 3-O-[β-D-galactopyranosyl(12→)]-α-L-arabinofuranosyl (1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,16α, 21β, 22α, 28-pentahydroxyolean-12-ene, also known as Xanifolia-Y1

The above compounds (Y and Y1) have anti-cancer effect. They inhibit the growth of human ovarian cancer (FIG. 3, 4).

These compounds (Y and Y1) are two of the active components identified from extracts of Xanthoceras sorbifolia by methods of FPLC and HPLC as shown in FIG. 5, 6, 7.

The compound Y is purified with procedure as described in this application (FIG. 7A). The purified compound Y shows 10 times higher potency (IC50=1.5 ug/ml) than the original extract (IC50=25 ug/ml) (FIG. 8). The compound Y has a high selectivity toward ovarian cancer (FIG. 9).

The compound Y shows inhibitory activity toward the following human cancer cells (eleven human cancer cell lines were tested in this study) with a higher potency toward ovarian carcinoma (comparison of activities is presented in FIG. 10 and Table 3.1).

This invention provides the extract of Xanthoceras Sorbifolia against cancer growth. The cancer includes, but is not limited to ovary cancer, bladder cancer, prostate cancer, leukocytes cancer, and bone cancer.

The compounds can be isolated from the plant called Xanthoceras Sorbifolia or can be synthesized chemically, or extracted from other biological sources.

This invention is related to the use of extracts of Wenguanguo. Extracts from husks, leaves, branches or stems, and fruit-stems, roots and barks of the Wenguanguo can be combined and this invention discloses methods of their preparation.

The extracts contain saponins, saccharides, proteins, glycosides, flavonoids, curmarin extracts, alkaloid extracts, organic acid extracts, tannin and others.

This invention provides the extract of Xanthoceras Sorbifolia for preventing cerebral aging; for improving memory; for improving cerebral functions; for curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence, Alzheimer's disease, brain trauma, or other diseases caused by cerebral dysfunctions.

Wenguanguo extracts may be used for accelerating the growth of bladder, for suppressing deep sleep, for increasing alertness in a sleeping subject, for modulating the release, breakdown and uptake of Antidieuretic hormone (ADH) and its receptors, for modulating the secretion, breakdown and uptake of Adrenocorticotropic hormone (ACTH) and its receptors, for modulating the release, breakdown and uptake of 5-hydroxytryptamine and its receptors, for modulating the release, breakdown and uptake of Acetycholine (Ach) and its receptors, for modulating the release, breakdown and uptake of Adrenaline (AD) and its receptors, for modulating the release, breakdown and uptake of Dopamine (DA) and its receptors, for modulating the release, breakdown and uptake of Norepinephrine (NE) and its receptors, for preventing sleep paralysis, for modulating the formation, release, breakdown and activity of neuropeptides and their receptors, for curing cancer, including but not limited to breast cancer, leukocyte cancer, liver cancer, ovary cancer, bladder cancer, prostate cancer and brain cancer, and for improving the functions of the lung and the bladder.

This invention provides a compound comprising a sugar, a triterpene or Sapogenin, and side chain at Carbon 21 and/or 22 including Angeloyl groups, operatively linked to form a biologically active compound. In an embodiment, the compound comprises one or more sugars.

This invention provides a salt of the above-described compounds.

This invention provides a pharmaceutical composition comprising an effective amount of the above-described compounds and a pharmaceutically acceptable carrier(s).

This invention provides a method for isolating compounds from Xanthoceras Sorbifolia comprising steps of: extracting Xanthoceras Sorbifolia powder with an appropriate amount of one or more organic solvents for an appropriate amount of time to form an organic extract; collecting the organic extract; refluxing the organic extract to form a second extract; removing the organic solvent from the second extract; drying and sterilizing the second extract to form a Xanthoceras Sorbifolia extract powder; fractionating the extract powder to obtain one or more components of the extract powder; identifying the bioactive components of the extract powder; purifying one or more bioactive components of the extract powder with FPLC to obtain one or more fraction of the bioactive component; and isolating the pure compound with preparative HPLC.

This invention provides a compound having a Structure verified by NMR spectral data derived from proton NMR, Carbon NMR, 2D NMR of the Heteronuclear Multiple Quantum Correlation (HMQC), Heteronuclear Multiple Bond Correlation (HMBC), and COSY, and Mass spectral data derived from WADLI-TOF and ESI-MS.

This invention provides the chemical features of a compound and its derivatives which are effective against cancer. The compounds or compositions of the present invention regulate the receptors or components of cell such as G-protein receptor, Fas protein, receptor Tyrosine Kinases, Mitogen, mitogen receptor. The compound inhibits cellular pathways include TGF Beta-smad, FGF, TGF-beta and TGF-alphaa, ras-GTPase-MAP kinase, jun-fos, Src-fyn, Jak-Jnk-STAT, BMP, Wnt, myc-cell proliferation, etc. The mutation of cancer cell causes the cell-death program to become inactive, allowing cells to divide indefinitely. The Xanthoceras Sorbifolia derived compound and/or composition regulates the components and receptors and re-activates the cell death program.

Abnormal changes in components' activities in pathways cause the cells to fail to stop proliferating so as to form cancer. The pathways include TGF Beta-smad, FGF, TGF-beta and TGF-alphaa, ras-GTPase-MAP kinase, jun-fos, Src-fyn, Jak-Jnk-STAT, BMP, Wnt, myc-cell proliferation, etc. The mutation of cancer cell causes the cell-death program to become inactive, allowing cells to divide indefinitely. The Xanthoceras Sorbifolia derived compound and/or composition regulates the components and receptors and re-activates the cell death program.

In accordance with these and other objects of the invention, a brief summary of the present invention is presented. Some simplifications and omission may be made in the following summary, which is intended to highlight and introduce some aspects of the present invention, but not to limit its scope. Detailed descriptions of a preferred exemplary embodiment adequate to allow those of ordinary skill in the art to make and use the inventive concepts will follow in later sections.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 shows the structure Compound Y with the formula of C₅₇H₈₈O₂₃ and the chemical name of 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β, 15α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene.

FIG. 2 shows the structure of Compound Y1 with the formula of C₆₅H₁₀₀O₂₇ and the chemical name of 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl (1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,16α, 21β, 22α, 28-pentahydroxyolean-12-ene.

FIG. 3 shows the anticancer activity of purified Compound Y. The periment was performed on ovarian cancer cells (OCAR-3) and the inhibition activity was determined by MTT assay (for details, refer to Experiment 3). Abscissa: Concentration (ug/ml). Ordinate: % Cell Growth. The IC50 is approximately 1 ug/ml. A: Point scale. B: Linear scale.

FIG. 4 shows the inhibition of the purified Compound Y1 and Compound Y2 on Ovarian cancer cells' growth.

FIG. 5 shows the results of the screening of cell growth activity of fractions obtained after FPLC chromatography. The assay was conducted with bladder cells. The fractions obtained from FPLC as shown in FIG. 20 were used. As shown in this figure, that different components of Xanthoceras Sorbifolia extracts cause either growth or inhibition effects on cells. Only fraction 5962 (Fraction Y) causes cell inhibition. Fractions 610 and 1116 cause minor stimulation of cell growth. Abscissa: concentration (ug/ml). Ordinate: % Cell Growth (determined by MTT assay).

FIG. 6 shows the separation of the components of Xanthoceras Sorbifolia extract by HPLC with a μbondapak C18 column. Details of experiment was presented in Experiment 2.

FIG. 7 shows HPLC profile of Fraction Y with 45% Acetonitrile isocratic elution in a preparative C18 column (Delta Pak C18). Under these conditions, fractions Y, Y1 and Y2 are well separated from each other and they are collected individually.

FIG. 7A shows the purity of the collected Compound Y by HPLC using 45% acetonitrile isocratic elution in a preparative C18 column.

FIG. 8 shows the a growth curve of ovarian cancer cells after treatment with the crude extract of Xanthoceras Sorbifolia as determined by MTT assay. This is a preliminary study on the sensitivity of extract of Xanthoceras Sorbifolia on cancer cell. Cell lines from 11 different human organs were employed. With the crude extract, this figure shows the most sensitive cancer cells are Ovary cancer cells. Activities on other cancer cells were represented in FIGS. 10A-D.

FIG. 9 shows the comparison of potency of Compound Y between ovarian cancer cells and cervical cancer cells. Ovarian cancer cells are much more sensitive to Compound Y than the cervical cancer cells. The IC50 for Compound Y in ovary cells is about 1.5. This result confirms that the activity of compound Y is more selective toward ovary cancer.

FIGS. 10A-D show the growth curves of cancer cells derived from different human organs as determined by MTT assay. After treatment with the extract of Xanthoceras Sorbifolia, growth curves of different cell lines were presented and their sensitivities (base on IC50 values) were determined.

-   10A: Sensitive: bladder and bone. -   10B: Semi-sensitive: leukocyte and liver. -   10C: Marginal sensitive: prostate, breast and brain. -   10D: Least sensitive: colon, cervix and lung.

FIG. 11 shows the sprectrum of proton NMR of Compound Y.

FIG. 12 shows 2D NMR (HMQC) results of Compound Y. Also see Table 5.2 for the listed chemical shift data.

FIG. 13 shows 2D NMR (HMBC) results of Compound Y. Also see Table 5.3 for the listed chemical shift data.

FIG. 14 shows the Mass spectrum of compound Y with MALDI-TOF (high mass): Y+Matrix (CHCA)+Angiotensin 1 “two point calibration”.

FIG. 15 shows the Mass spectrum of compound Y with ESI-MS.

FIG. 16 shows the Proton NMR spectrum of Compound Y1.

FIG. 17 shows the 2D NMR (HMQC) results of Compound Y1. Also see the chemical shift data from Table 6.2.

FIG. 18 shows the 2D NMR (HMBC) results of Y1. Also see the chemical shift data from Table 6.3.

FIG. 19 shows COSY-NMR profile of Y1 with chemical shift data from Table 6.4.

FIG. 20 shows the elution profile of an extract of Xanthoceras Sorbifolia in FPLC with 10-80% gradient. Ordinate: Optical density (at 245 nm). Abscissa: Fractions (5 ml/fraction).

FIG. 21 shows the Proton-NMR spectra of compound R1.

FIG. 22 shows the 2D NMR (HMQC) spectra of compound R1.

FIG. 23 shows the 2D NMR (HMBC) spectra of compound R1.

FIG. 24 shows the 2D NMR (COSY) spectra of compound R1.

FIG. 25 shows the C13 NMR spectra of compound R1.

FIG. 26 shows the chemical structure of Compound R1: 3-O-[angeloyl-(1→3)-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β,21β,22α,28-tetrahydroxyolean-12-ene

FIG. 27 shows the chemical structure of Compound O54.

FIG. 28 shows the Proton-NMR spectra of compound O54.

FIG. 29 shows the 2D NMR (HMQC) spectra of compound O54.

FIG. 30 shows the 2D NMR (HMBC) spectra of compound O54.

FIG. 31 shows one of the four possible chemical structures of Y1. A: structure Y1-1.

FIG. 32 shows one of the four possible chemical structures of Y1. B: structure Y1-2.

FIG. 33 shows one of the four possible chemical structures of Y1. C: structure Y1-3.

FIG. 34 shows one of the four possible chemical structures of Y1. D: structure Y1-4. R1=A or B or C; R2=A or B or C; R3=A or B or C.

R1 R2 R3 1 A A A 2 A A B 3 A A C 4 A B A 5 A B B 6 A B C 7 A C A 8 A C B 9 A C C 10 B A A 11 B A B 12 B A C 13 B B A 14 B B B 15 B B C 16 B C A 17 B C B 18 B C C 19 C A A 20 C A B 21 C A C 22 C B A 23 C B B 24 C B C 25 C C A 26 C C B 27 C C C

FIG. 35 shows the chemical structure of Y-a.

R5=B or C or S1 (see note 1); R1=A or B or C; R4=B or C; Note 1: A=angeloy, B=acetyl, C=H, S1=chain with one or more sugar such as D-glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D-glucuronic acid, D-galacturonic acid and their derivatives.

FIG. 36 shows the chemical structure of Y-b.

R5=B or C or S1 (see note 1); R1=A or B or C; R4=B or C; Note 1: A=angeloy, B=acetyl, C=H, S1=chain with one or more sugar such as D-glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D-glucuronic acid, D-galacturonic acid and their derivatives.

FIG. 37 shows the chemical structure of Y-c.

FIG. 38 shows the chemical structure of Y1-a.

R5=B or C or S1 (see note 1); R1=A or B or C; R4=B or C; Note 1: A=angeloy, B=acetyl, C=H, S1=chain with one or more sugar such as D-glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D-glucuronic acid, D-galacturonic acid and their derivatives.

FIG. 39 shows the chemical structure of Y1-b.

R5=B or C or S1 (see note 1); R1=A or B or C; R4=B or C; Note 1: A=angeloy, B=acetyl, C=H, S1=chain with one or more sugar such as D-glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D-glucuronic acid, D-galacturonic acid and their derivatives.

FIG. 40 shows the chemical structure of Y1-c.

R5=B or C or S1 (see note 1); R1=A or B or C; R4=B or C; Note 1: A=angeloy, B=acetyl, C=H, S1=chain with one or more sugar such as D-glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D-glucuronic acid, D-galacturonic acid and their derivatives.

FIG. 41 shows the absorption spectrum of Xanthoceras Sorbifolia extract. Abscissa: Wavelength in nm. Ordinate: Optical Density. The extract has three absorption maximum at 207 nm, 278 nm and 500 nm.

FIG. 42 shows elution profile of Fraction 5962 with 64% acetonitrile isocratic elution. Two major FPLC fractions X and Y are separated. Ordinate: optical density (254 nm). Abscissa: fraction Number (1 ml/fraction).

FIG. 43 shows the comparison of inhibition activity in bladder cells by Fractions X (2021) and Y (2728). Only Fraction Y has inhibition activity.

FIG. 44 shows HPLC profile of Fraction Y with 45% Acetonitrile isocratic elution. Two major and 2-3 minor compounds were identified.

FIG. 45 shows purification of Fraction R from with FPLC.

FIG. 46 shows the HPLC analysis of fractions #9, #10 and #11 obtained from FPLC.

FIG. 47 shows purification of component-R with HPLC (Delta-Pak C18). A: Extract from fraction #10 of FPLC (iso-30) was further separated by HPLC. B: Rechromatogram of the major component under same condition as described in A.

FIG. 48. Fractionation of Fraction-O from FPLC with HPLC with 20% acetonitrile isocratic elution (iso-20).

FIG. 49. Rechromatography of O28 and O34 (from iso-20).

FIG. 50. Rechromatography of O54 (from iso-20).

FIG. 51 shows the proton NMR spectrum of Y2.

FIG. 52 shows the 2D NMR spectrum of Y2 (HMQC).

FIG. 53 shows the proton NMR spectrum of Y4.

FIG. 54 shows the 2D NMR (HMQC) spectrum of Y4.

FIG. 55 shows the proton NMR spectrum of O28.

FIG. 56 shows the 2D NMR (HMQC).

FIG. 57 shows the proton NMR spectrum of O34.

FIG. 58 shows the 2D NMR (HMQC) spectrum of O34.

FIG. 59 shows the effects of the extract X and Y on the quantity of urine in mice after 10 days of administration of X and Y.

FIGS. 60(a) and (b) show the water maze learning effect of plant extract administration of aging mice for 9 days.

FIGS. 61(a) and (b) show the result of water maze learning of 3 days injected pentobarbital.

FIG. 62 shows the sleep cycle of a typical person.

FIG. 63. Table 15A-1 shows results of urine volume with water load after administration extract for 25 days. FIG. 63A shows the urine volume with water load after administration of FS(X) and FS(Y) extract for 25 days.

FIG. 64. Table 15A-2 shows results of discharging urine speed with water load after administration extract for 25 days. FIG. 64A shows the discharging urine speed with water load after administration of FS(X) and FS(Y) extract for 25 days.

FIG. 65. Table 15A-3 shows results of urine specific gravity and pH with water load after administration extract for 25 days.

FIG. 66. Table 15A-4 shows concentration of Na+, K+ and Cl− in urine with water load after administration extract for 25 days.

FIG. 67 shows the structure of the compound.

FIG. 68 shows the structure of the compound.

FIG. 69 shows the structure of the compound.

FIG. 70 shows the structure of the compound.

FIG. 71 shows the structure of the compound.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a compound comprising the following structure, with the formula of C₅₇H₈₈O₂₃ and the name of 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β, 15α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene, also known as Xanifolia-Y This compound was isolated from Xanthoceras sorbifolia

This compound belongs to saponins consist of a triterpene, sugar moiety and angeloyl groups links to the backbone. The angeloyl groups linked to the C21 and C22 positions. This compound has the anti-cancer activity.

The assignment of this structure is supported by the spectral data (1 D H-NMR, C-NMR, 2D NMR (HMBC, HMQC, COSY), and MS (MALDI-TOF, EMS). Accordingly, this compound has the characteristic property as shown in FIGS. 11-15 or Table 5.1.

This invention provides another compound comprising the following structure, with the formula of C₆₅H₁₀₀O₂₇ and the name of 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl (1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,16α, 21β, 22α, 28-pentahydroxyolean-12-ene, also known as Xanifolia-Y1

This compound belongs to saponins consist of a triterpene, sugar moiety connected to the backbone. A sugar that linked to the C21 position has two angeloyl groups attached. This compound has anti-cancer activity.

The assignment of this structure is supported by the spectral data (1 D H-NMR, C-NMR, 2D NMR (HMBC, HMQC, COSY), and MS (MALDI-TOF, EMS). Accordingly, this compound has the characteristic property as shown in FIGS. 16-19 or Table 6.1.

This invention provides evidence to show that the extract of Xanthoceras Sorbifolia contains anticancer activity. The experiments for determining the anti-cancer activity employed human cells lines derived from eleven human organs (HTB-9 (bladder), HeLa-S3 (cervix), DU145 (prostate), H460 (lung), MCF-7 (breast), K562 (leukocytes), HCT116 (colon), HepG2 (liver), U2OS (bone), T98G (brain) and OVCAR-3 (ovary)). Among the 11 cell lines studies, their sensitivity toward Xanthoceras Sorbifolia extract can be divided into four groups: (A) most sensitive: Ovary (FIG. 8); (B) Sensitive: bladder, bone, prostate, and leukocyte, (C) marginal sensitive: liver, breast, and brain; and (D) lease sensitive: colon, cervix, and lung. (FIG. 10A-D). Their IC50 values are listed in Table 3.1.

TABLE 3.1 IC50 values of Xanthoceras Sorbifolia Extract Determined in Different Cancer Cells IC50 determined by MTT assay Cancer cells from different organs (ug/ml) Ovary (most sensitive) 15-15 Bladder (sensitive) 45-50 Bone 40-55 Prostate 40-50 Leukocyte 45-50 Liver (marginal sensitive) 45-65 Breast 65 Brain 70-85 Colon (least sensitive) 90 Cervix 115 Lung 110

In order to identify the active compounds of Xanthoceras Sorbifolia, the extract from Xanthoceras Sorbifolia were separated by chromatography comprising FPLC (Fast Protein Liquid Chromatography) and HPLC (High Preferment Liquid Chromatography). Multiple fractions were obtained by FPLC procedures (FIG. 20) and HPLC (FIG. 6).

Analysis of the components of Xanthoceras Sorbifolia by HPLC shows that the extract comprises 26 identifiable fractions (named a to z) as shown in FIG. 6.

Anti-cancer activities of these fractions were determined by the MTT assay. Only fraction Ys has the anti-cancer activity (FIG. 5). Fraction Ys were further separated into 4 components (FIG. 7). The compounds Y and Y1 are the active components currently isolated from Xanthoceras Sorbifolia as shown in FIG. 3-4.

The invention tested the inhibition effects of ovarian cancer cells with the MTT assay, and the compound Y shows 10 times higher potency (IC50=1.5 ug/ml) (FIG. 3) than the original crude extract as shown in FIG. 8 (IC50=25 ug/ml).

The selectivity of compound Y was tested, and it has been found that compound Y has a much higher potency toward ovarian cancer cells as compared to the cervical cancer cells (FIG. 9).

The compounds Y1 and Y2 have anti-cancer activity as shown in FIG. 4.

This invention provides the detail isolation procedures for the active compounds of the present invention.

This invention provides the spectral data evidence (1D H-NMR, C-NMR, 2D NMR (HMBC, HMQC, COSY), and MS (MALDI-TOF, ESI-MS) in supporting the assigned structures.

This invention provides a salt of the above-described compounds.

This invention provides a composition comprising the above-described compounds and a suitable carrier.

This invention provides a pharmaceutical composition comprising an effective amount of the above-described compounds and a pharmaceutically acceptable carrier.

This invention provides an anti-ovarian cancer agents and composition comprising the above-described composition.

This invention provides the compositions against cancer growth. The cancer includes, but is not limited to bladder cancer, bone cancer, and ovary cancer.

This invention provides a composition comprising the above compounds and their derivatives for inhibition of tumour growth.

The following methods and materials were used in the examples and/or experiments described in this application.

Cells.

Human cancer cell lines were obtained from American Type Culture Collection: HTB-9 (bladder), HeLa-S3 (cervix), DU145 (prostate), H460 (lung), MCF-7 (breast), K562 (leukocytes), HCT116 (colon), HepG2 (liver), U2OS (bone), T98G (brain) and OVCAR-3 (ovary). Cells were grown in culture medium (HeLa-S3, DU145, MCF-7, Hep-G2 and T98G in MEN (Earle's salts); HTB-9, H460, K562, OVCAR-3 in RPMI-1640; HCT-116, U2OS in McCoy-5A) supplemented with 10% fetal calf serum, glutamine and antibiotics in a 5% CO2 humidified incubator at 37° C.

MTT Assay.

The procedure for MTT assay followed the method described in (Carmichael et al., 1987) with only minor modifications. Cells were seeded into a 96-wells plate at concentrations of 10,000/well (HTB-9, HeLa, H460, HCT116, T98G, OVCAR-3), 15,000/well (DU145, MCF-7, HepG2, U205), or 40,000/well (K562), for 24 hours before drug-treatment. Cells were then exposed to drugs for 48 hours (72 hours for HepG2, U205, and 96 hours for MCF-7). After the drug-treatment, MTT (0.5 mg/ml) was added to cultures for an hour. The formation of formazan (product of the reduction of tetrazolium by viable cells) was dissolved with DMSO and the O.D. at 490 nm was measured by an ELISA reader. The MTT level of cells before drug-treatment was also measured (T0). The % cell-growth (% G) is calculated as:

% G=(TD−T0/TC−T0)×100  (1),

where TC or TD represent O.D. readings of control or drug-treated cells. When T0>TD, then the cytotoxicity (LC) expressed as % of the control is calculated as:

% LC=(TD−T0/T0)×100  (2).

In addition to the compounds Y and Y1, other compounds from the extract including R1 and O54, were also purified and the structure were determined by 1D H-NMR, C13-NMR, 2D NMR (HMQC, HMBC, COSY); MS (MALDI-TOF).

The Structure of Compound R1 shown below and in FIG. 26, has a chemical formula of C₆₅H₁₀₆O₂₉ and chemical name of

3-O-[angeloyl-(1→3)-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)β-D-glucopyranosyl-(1→6)β-D-glucopyranosyl-3β, 21β, 22α, 28-tetrahydroxyolean-12-ene, also known as Xanifolia-R1.

The assignment of this structure is supported by the spectral data (1 D H-NMR, C-NMR, 2D NMR (HMBC, HMQC, COSY), and MS (MALDI-TOF, EMS). Accordingly, this compound has the characteristic property as shown in FIGS. 21-25 or Table 8.1

Compound-O54

This invention provides a compound O54 with formula of C₆₀H₁₀₀O₂₈ and the structure was determined by 1D NMR, 2D NMR, MS).

The Structure of Compound O54 (also shown in FIG. 27):

The name of Compound O54 is The chemical name of compound-O54 is: 3-O-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β, 21β, 22α, 28-tetrahydroxyolean-12-ene

The assignment of this structure is supported by the spectral data (1 D H-NMR, 2D NMR (HMBC, HMQC). Accordingly, this compound has the characteristic property as shown in FIGS. 28-30 and table 9.1.

In other embodiments, the structures of the compounds are as follows:

Structure 1 as shown in FIG. 31 Structure 2 as shown in FIG. 32 Structure 3 as shown in FIG. 33 Structure 4 as shown in FIG. 34 Structure Y-a as shown in FIG. 35 Structure Y-b as shown in FIG. 36 Structure Y-c as shown in FIG. 37 Structure Y1-a as shown in FIG. 38 Structure Y1-b as shown in FIG. 39 Structure Y1-c as shown in FIG. 40

This invention provides a compound comprising a sugar, a triterpene or Sapogenin, and a side chain at Carbon 21 and 22 or Angeloyl groups. In an embodiment, the compound comprises two or more sugars.

This invention provides a salt of the above-described compounds.

This invention provides a composition comprising the above-described compounds and a suitable carrier.

This invention provides a pharmaceutical composition comprising an effective amount of the above-described compounds and a pharmaceutically acceptable carrier.

This invention provides an anti-ovarian cancer agents and composition comprising the above-described composition.

This invention provides the compositions against cancer growth. The cancer includes, but is not limited to bladder cancer, bone cancer, and ovary cancer.

This invention provides composition comprising the above compounds and their derivatives to inhibit tumour growth.

This invention provides composition comprising the above compounds and their derivatives to cure human immunodeficiency virus (HIV) or Severe Acute Respiratory Syndrome (SARS) or flux disease or inhibit virus activities.

This invention provides medicine or health food for improving the sensory stretch receptor in the bladder wall, inhibiting AChE or use as an anti-inflammatory agent.

This invention provides a method for preventing cerebral aging, improving memory, improving cerebral functions and curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer's disease, autism, brain trauma, Parkinson's disease and other diseases caused by cerebral dysfunctions, and treating arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder and treating impotence and premature ejaculation.

This invention provide methods for inhibiting tumor cell growth or to treat patients with HIV or SARS, or inhibit virus activities, or for preventing cerebral aging, improving memory, improving cerebral functions and curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer's disease, autism, brain trauma, Parkinson's disease and other diseases caused by cerebral dysfunctions, and treating arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder and treating impotence and premature ejaculation. comprising contacting an amount of the compound is a triterpene or sapongenin with any two of angeloyl group or tigloyl group or senecioyl group or their combinations attach to carbon 21 and 22, or any two of angeloyl group or tigloyl group or senecioyl group or their combinations attached to a sugar moiety which bonds to carbon 21 or 22.

Wenguanguo is a species of the sapindaceae family. Its scientific name is Xanthoceras sorbifolia Bunge. Wenguanguo is the common Chinese name; others are Wenguannguo, Wenguanmu, Wenguanhua, and Xilacedeng. This plant can grow up to 8 meters in height. It features odd pinnately compound leaf, eraceme with white flowers, capsules with thick and woody husks. Wenguanguo is grown in Liaoning, Jilin, Hebei, Shandong, Jiangsu, Henan, Shanxi, Shaanxi, Gansu, Ningxia and Inner Mongolia, China. Its seeds are edible and have been used as a folk medicine to treat enuresis for centuries. Its branches and woods are also used as a folk medicine.

This invention is a further description of the extracts from Wenguanguo, their uses and methods for preparation. This invention provides the extracts that can prevent enuresis by improving patients' cerebral functions so that patients can be more aware of the signals sent from the bladder and wake up from deep sleep. When the bladder is full of urine, the smooth muscle of the bladder is extended, which produces a signal up to the cerebral cortex and cerebellum through the pelvic nerve and the sacral spinal cord. The response of the cerebral cortex and cerebellum to the signal is to make the bladder sustain contracted but the sphincter relaxed. The urine is then discharged. When the bladder is filled with urine via the urethra during sleep, the detrusor stretches, allowing the bladder to expand. As the bladder starts to accumulate urine, it will stimulate the stretch receptors in the bladder that will generate signals continually to the brain according to the amount of urine accumulated in the bladder. When the bladder is full enough with urine, then the intra-vesicle has accumulated enough pressure for the brain to recognize and wake the person to urinate. If the signal is not strong enough to wake the sleeping person or blocked due to impairment of cerebral function, then enuresis occurs. This particular plant extract can cure enuresis by improving cerebral functions.

The sensory stretch receptors are located within the bladder wall and help with assessing the degree of bladder fullness. This information is transmitted up to the spinal cord and then via the spinothalamic tracts to the central nervous system. The extracts of Wenguanguo make the central nervous system more aware of the signal.

When the bladder becomes contracted under stress and nervousness, the capacity of the urinary bladder will be reduced and then the frequent micturition occurs. The extracts of Wenguanguo can relax the bladder for storing more urine.

The capacity of the urinary bladder is reduced because of aging, and this may even happen to middle-aged people. They suffer from experience of early detrusor contraction due to a sense of urgency to empty the bladder at low urine level. The extracts of Wenguanguo can help relax the detrusor and therefore the bladder capacity increases and urinary frequency decreases.

Patients with detrusor overactivity, detrusor instability, detrusor hyper-reflexia or uninhibited bladder have early, forceful detrusor contractions before the bladder is full. This creates urgency and frequency urinary discharge. The extract of Wenguanguo relaxes the patient's detrusor. The bladder becomes stable and can store a full amount of urine.

The smooth muscle of the urinary bladder has two functions: When the bladder is relaxed, the urine is stored. When it is contracted, the urine will be discharged. The sensory stretch receptors are located within the bladder wall to assess the bladder's fullness. This information is transmitted up the spinal cord via the spinothalamic tracts to the nervous system. The brain generates inhibitory signals when detrusor relaxation is desired. But the brain generates excitatory signal when detrusor contraction is desired. The extracts of Wenguanguo can relax the bladder tissue by inhibiting Acetylcholinesterase, AchE. The inhibiting effect can be maintained for a long period of time. The extracts of Wenguanguo are a good AChE inhibitor that can cure the diseases caused by deficiency of Acetylcholine, ACh.

Antidiuretic hormone (ADH) is stored in the posterior pituitary gland in the brain. It is the primary regulator of body water. ADH acts on the kidneys to increase or decrease total body water. This has an effect on the volume of urine generated by the kidney. The release of ADH is controlled by the cells of osmoreceptors and baroreceptors. Osmoreceptors are the specialized cell hypothalamus. These cells sense the concentration of particles in the blood. When the concentration of particles is higher, more ADH will be released by the pituitary. This stimulates retention of water to dilute body fluids. When the concentration is lower, less ADH will be released by the pituitary. Baroreceptors are located in the right atria and great veins and carotid sinus the specialized area in the heart that sense blood volume and blood pressure. The heart will generate signals to the hypothalamus and pituitary to release more ADH when blood volume or blood pressure is low and vice versa. The extracts of Wenguanguo can regulate the release of ADH which will reduce the volume of urine produced by the body.

This invention relates to the flavone extracts from Wenguanguo husks and fruit-stems, and methods of their preparation. The methods for preparing the extracts from Wenguanguo husks and fruit-stems comprise the following steps: extracting Wenguanguo powder made from husk and fruit-stem with ethanol 3-4 times to form an ethanol extract; removing the ethanol from the ethanol extract to form an aqueous extracts; drying the aqueous extracts to form the flavone extracts that is yellow powder.

This invention provides a composition comprising extracts from husks and fruit-stems which are flavonols, flavanols, dihydroflavonols, phenoloids, and others.

This invention relates to the crude flavone extracts from Wenguanguo leaves that includes a water-soluble flavone extracts and a water-insoluble flavone extract and methods of their preparation. The methods for preparing the extracts from Wenguanguo leaves comprise the following steps: extracting Wenguanguo powder made from the leaves with ethanol for 3 times to form an ethanol extract; concentrating the ethanol extract to form a concentrated condensed extracts; extracting the concentrated extract with hot water to from an aqueous extracts and a water-insoluble extract; drying the aqueous extracts and the water-insoluble extract to form a water-soluble flavone extracts and a water-insoluble flavone extract. This invention provides a composition comprising the crude extracts from leaf which are flavonols, flavanols, dihydroflavonols, phenoloids and others.

This invention relates to the flavone extracts from Wenguanguo branches or stems and methods of their preparation. The methods for preparing the extract from branches or stem comprise the following steps: extracting Wenguanguo powder made from the branches or stems with ethanol for 4 times to form an ethanol extract; removing the ethanol from the ethanol extract to form an aqueous extracts; drying the aqueous extracts to form flavone extracts which is a yellowish powder.

This invention provides a composition comprising extracts from Wenguanguo branches and stems which are flavonols, flavanols, dihydroflavonols, phenoloids and others.

This invention relates to the flavone extracts from Wenguanguo kernels and methods of their preparation. The methods for preparing the extract from kernels comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and drying them to form the kernel powder; extracting the kernel powder with ethanol to form an ethanol extract; removing the ethanol from the ethanol extract to form an aqueous extract; drying the aqueous extracts to form a flavone extracts that is a yellow powder.

This invention provides a composition comprising extracts from kernel which are flavonols, flavanols, dihydroflavonols, proteins, phenoloids, and others.

This invention relates to the flavone extract from Wenguanguo root, and methods of their preparation. The methods for preparing the flavone extract from Wenguanguo root comprise the following steps: extracting Wenguanguo powder made from root with ethanol 3-4 times to form an ethanol extract; removing the ethanol from the ethanol extract to form an aqueous extract; drying the aqueous extracts to form the flavone extracts which is a yellow powder.

This invention provides a composition comprising extracts from roots of Wenguanguo which are flavonols, flavanols, dihydroflavonols, phenoloids and others.

This invention relates to the flavone extracts from Wenguanguo barks, and methods of their preparation. The methods for preparing the bark extracts from Wenguanguo barks comprise the following steps: extracting Wenguanguo powder made from the barks with ethanol 3-4 times to form an ethanol extract; removing the ethanol from the ethanol extract to form an aqueous extract; drying the aqueous extracts to form the flavone extracts which is a yellowish powder.

This invention provides an extract composition from Wenguanguo barks comprising flavonols, flavanols, dihydroflavonols, phenoloids and others.

This invention is related to the combined extracts from Wenguanguo husks or fruit-stems and method of their preparation. The methods for preparing the extract from the husks or fruit-stems comprise the following steps: extracting Wenguanguo powder made from the husks or fruit-stems with an organic solvent (ethanol, methanol and others) to form an organic extract; removing the organic solvent from the organic extract to from an aqueous extracts; drying and sterilizing the aqueous extracts to form the combined extracts.

This invention provides a composition comprising the combined extracts from the husks or fruit-stems of the Wenguanguo. The combined extracts comprise saponins, saccharides, proteins and others.

This invention is related to the combined extracts from Wenguanguo leaves and method of their preparation. The methods for preparing the extracts from the leaves comprise the following steps: extracting Wenguanguo powder made from leaves with an organic solvent (ethanol, methanol and others) to form an organic extract; removing the organic solvent from the second extract to an aqueous extract; extracting the aqueous extract with ether and water to form an second aqueous extract; extracting the second aqueous extract with n-butanol to form a n-butanol extract; removing the n-butanol from the n-butanol extract to form a third aqueous extract; drying and sterilizing the third aqueous extract to form the combined extracts.

This invention provides a composition comprising the organic extracts from the leaves of the Wenguanguo. The organic extracts comprise saponins, saccharides, proteins and others.

This invention is related to the combined extracts from Wenguanguo branches or stems and method of their preparation. The methods for preparing the extracts from the branches or stems comprise the following steps: extracting Wenguanguo powder made from the branches or stems with an organic solvent (ethanol, methanol and others) to form an organic extract; removing the organic solvent from the second extract to an aqueous extract; drying and sterilizing the aqueous extracts to form the combined extracts.

This invention provides a composition comprising the organic extracts from the branches, and stems and of the Wenguanguo. The organic extracts comprise saponins, saccharides, proteins and others.

This invention is related to the combined extracts from Wenguanguo kernels and method of their preparation. The methods for preparing the extracts from Wenguanguo kernels comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and drying them to form the kernel powder; extracting the kernel powder with an organic solvent (ethanol, methanol and others) to form an organic extract; removing the organic solvent from the second extract to an aqueous extract; drying and sterilizing the aqueous extracts to form the combined extracts.

This invention provides a composition comprising the organic extracts from the kernels of the Wenguanguo. The combined extracts comprise saponins, saccharides, proteins and others.

This invention is related to the combined extracts from Wenguanguo roots and method of their preparation. The methods for preparing the extracts from Wenguanguo roots comprise the following steps: extracting Wenguanguo powder made from the roots with an organic solvent (ethanol, methanol and others) to form an organic extract; removing the organic solvent from the organic extract to from an aqueous extracts; drying and sterilizing the aqueous extracts to form the combined extracts.

This invention provides a composition comprising the combined extracts from the roots of the Wenguanguo. The combined extracts comprise saponins, saccharides, proteins and others.

This invention is related to the combined extracts from Wenguanguo barks and method of their preparation. The methods for preparing the extracts from the barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder made from the barks with an organic solvent (ethanol, methanol and others) to form an organic extract; removing the organic solvent from the organic extract to from an aqueous extract; drying and sterilizing the aqueous extracts to form the combined extracts.

This invention provides a composition comprising the combined extracts from the barks of the Wenguanguo. The combined extracts comprise saponins, saccharides, proteins and others.

This invention provides the crude saponins from the husks or fruit-stems or seed's shell of Wenguanguo. The methods for preparing the crude saponins from Wenguanguo husks or fruit-stems comprise the following steps: extracting Wenguanguo powder of the husks or fruit-stems with an organic solvent (ethanol, methanol and others) at ratio of 1:2 for 4-5 times, 20-35 hours for each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extract comprises saponins.

This invention provides the crude saponins from the leaves of Wenguanguo and method for their preparation. The methods for preparing the crude saponins from the leaves comprise the following steps: extracting Wenguanguo powder of the leaves with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form a second extract; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extract comprises saponins.

This invention provides the crude saponins from the branches and stems of Wenguanguo. The methods for preparing the crude saponins from the branches or stems comprise the following steps: extracting Wenguanguo powder of the branches or stems with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extract comprises saponins.

This invention provides the crude saponins from the kernels of Wenguanguo. The methods for preparing the crude saponins from Wenguanguo kernels comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract for 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; Extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extracts comprise saponins.

This invention provides the crude saponins from the roots of Wenguanguo and method for their preparation. The methods for preparing the crude saponins from Wenguanguo roots comprise the following steps: extracting Wenguanguo powder of the roots with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extracts contain saponins.

This invention provides the crude saponins from the barks of Wenguanguo and method for their preparation. The methods for preparing the crude saponins from the barks comprise the following steps: extracting Wenguanguo powder of the barks with an organic solvent (ethanol, methanol and others) at a ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extracts comprise saponins.

This invention provides a process of producing a coumarin extract from the husks or fruit-stems of Wenguanguo and their applications. The methods for preparing the coumarin extracts from husks or fruit-stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the husks or fruit-stems with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extracts from the husks or fruit-stems of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing a coumarin extract from the leaves of Wenguanguo and their applications. The methods for preparing the coumarin extracts from leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extracts from the leaves of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing a coumarin extract from the branches and stems of Wenguanguo and their applications. The methods for preparing the coumarin extract from the branches or stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder branches or stems with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the extract comprising crude coumarins.

This invention provides a composition comprising the coumarin extracts from the branches and stems of Wenguanguo. The extract comprises coumarins, coumaric glycosides, saccharides, proteins and others.

This invention provides the crude saponins from the leaves of Wenguanguo and method for their preparation. The methods for preparing the crude saponins from the leaves comprise the following steps: extracting Wenguanguo powder of the leaves with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form a second extract; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extract comprises saponins.

This invention provides the crude saponins from the branches and stems of Wenguanguo. The methods for preparing the crude saponins from the branches or stems comprise the following steps: extracting Wenguanguo powder of the branches or stems with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extract comprises saponins.

This invention provides the crude saponins from the kernels of Wenguanguo. The methods for preparing the crude saponins from Wenguanguo kernels comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract for 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; Extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extracts comprise saponins.

This invention provides the crude saponins from the roots of Wenguanguo and method for their preparation. The methods for preparing the crude saponins from Wenguanguo roots comprise the following steps: extracting Wenguanguo powder of the roots with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extracts contain saponins.

This invention provides the crude saponins from the barks of Wenguanguo and method for their preparation. The methods for preparing the crude saponins from the barks comprise the following steps: extracting Wenguanguo powder of the barks with an organic solvent (ethanol, methanol and others) at a ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extracts comprise saponins.

This invention provides a process of producing a coumarin extract from the husks or fruit-stems of Wenguanguo and their applications. The methods for preparing the coumarin extracts from husks or fruit-stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the husks or fruit-stems with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extracts from the husks or fruit-stems of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing a coumarin extract from the leaves of Wenguanguo and their applications. The methods for preparing the coumarin extracts from leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extracts from the leaves of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing a coumarin extract from the branches and stems of Wenguanguo and their applications. The methods for preparing the coumarin extract from the branches or stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder branches or stems with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the extract comprising crude coumarins.

This invention provides a composition comprising the coumarin extracts from the branches and stems of Wenguanguo. The extract comprises coumarins, coumaric glycosides, saccharides, proteins and others.

This invention provides a process of producing a coumarin extract from the kernels of Wenguanguo and their applications. The methods for preparing the coumarin extracts from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and drying them to form the kernel powder; extracting the kernel powder with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form an ether extract; neutralizing the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extracts from the kernels of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing a coumarin extract from the roots of Wenguanguo and their applications. The methods for preparing the coumarin extract from roots of Wenguanguo comprise the following steps: extracting

Wenguanguo powder of the root with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extracts from the roots of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing a coumarin extract from the barks of Wenguanguo and their applications. The methods for preparing the coumarin extract from barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the bark with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extract from the barks of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing an aqueous extract from the husks or fruit-stems of Wenguanguo and their applications. The method for preparing the water extracts from the husks or fruit-stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the husk or fruit-stem with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from the husks or fruit-stems of Wenguanguo The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the leaves of Wenguanguo and their applications. The method for preparing the water extracts from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from leaves of Wenguanguo. The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the branches or stems of Wenguanguo and their applications. The method for preparing the water extracts from branches or stems of Wenguanguo comprise the following steps: extracting the Wenguanguo powder of the branches or stems with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrating the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from the branches or stems of Wenguanguo. The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the kernels of Wenguanguo and their applications. The method for preparing the water extracts from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from kernels of Wenguanguo. The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the roots of Wenguanguo and their applications. The method for preparing the water extracts from the roots of Wenguanguo comprises the following steps: extracting Wenguanguo powder of the roots with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrating the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from the roots of Wenguanguo The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the barks of Wenguanguo and their applications. The method for preparing the water extracts from the barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the barks with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extracts from the barks of Wenguanguo The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an alkaloid extract from the husks of Wenguanguo and their applications. The methods for preparing the alkaloid extracts from the husks and fruit-stems of Wenguanguo comprising the following steps: extracting Wenguanguo powder of the husks or fruit-stems with water at a ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extract the alkalified aqueous extract by toluol to form a toluol extract; the toluol extract flows through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression a to form crude alkaloids.

This invention provides a composition comprising the alkaloid extract from the husks or fruit-stems of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the leaves of Wenguanguo and their applications. The methods for preparing the alkaloid extract from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with water at a ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collecting and alkalifying the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extracting the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression to form the alkaloid extract.

This invention provides a composition comprising the alkaloid extract from the leaves of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the branches and stems of Wenguanguo and their applications. The methods for preparing the extracts containing alkaloids from branches or stems of Wenguanguo comprising the following steps: extracting Wenguanguo powder of the branches or stems with water at ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extracting the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression to form the alkaloid extract.

This invention provides a composition comprising the extract containing crude alkaloids from the branches or stems of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the kernels of Wenguanguo and their applications. The methods for preparing the alkaloid extract from kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grounding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with water at ratio of 1:6 for 3-4 times, 10-15 hours for each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extract the alkalified aqueous extract by toluol to form a toluol extract; the toluol extract flows through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression to form the alkaloid extract.

This invention provides a composition comprising the alkaloid extract from the kernels of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the roots of Wenguanguo and their applications. The methods for preparing the alkaloid extract from the roots of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the Wenguanguo roots with water at a ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collecting and alkalifying the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extracting the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression a to form crude alkaloids.

This invention provides a composition comprising the alkaloid extract from the roots of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the barks of Wenguanguo and their applications. The methods for preparing the alkaloid extract from the barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the barks with water at ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extract the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression a to form crude alkaloids.

This invention provides a composition comprising the alkaloid extract from the barks of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing extract containing organic acids from husks and fruit-stems and their applications. The methods for preparing the extracts containing organic acids from the husks or fruit-stems of Wenguanguo comprise the following steps: extract Wenguanguo powder of the husks and or fruit-stems with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO³ extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising crude organic acids from the husks of Wenguanguo. The extract comprising aromatic organic acids, fatty organic acids, terpenoid organic acids and others

This invention provides a process of producing extract contains organic acids from leaf and their applications. The methods for preparing the extracts containing organic acids from the leaves of Wenguanguo comprise the following steps: extract Wenguanguo powder of the leaves with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO₃ solution to form a NaHCO₃ extract; acidize and filter the NaHCO₃ extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising the extract comprising crude organic acids extract from the leaves of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract contains organic acids from branches and stems and their applications. The methods for preparing the extracts comprising organic acids from the branches or stems of Wenguanguo comprise the following steps: extract Wenguanguo powder of the branches or stems with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO₃ solution to form a NaHCO₃ extract; acidize and filter the NaHCO₃ extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising the crude organic acids extract from the branches and stems of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract comprise organic acids from kernels and their applications. The methods for preparing the extracts comprising organic acids from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grounding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO₃ solution to form a NaHCO₃ extract; acidize and filter the NaHCO₃ extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising crude organic acids extract from the kernels of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract contains organic acids from the roots of Wenguanguo and their applications. The methods for preparing the extracts containing organic acids from the roots of Wenguanguo comprise the following steps: extract Wenguanguo powder of the roots with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO₃ solution to form a NaHCO₃ extract; acidize and filter the NaHCO₃ extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising the extract comprising crude organic acids from the roots of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract comprising organic acids from barks of Wenguanguo and their applications. The methods for preparing the extracts containing organic acids from the barks of Wenguanguo comprise the following steps: extract Wenguanguo powder of the bark with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO₃ solution to form a NaHCO₃ extract; acidize and filter the NaHCO₃ extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising the extract comprising crude organic acids from the barks of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides two methods of producing a tannin extract from Wenguanguo husks and fruit-stems and its usage. The first method for preparing the tannin extract from the husks or fruit-stems of Wenguanguo comprises the following steps: extracting Wenguanguo powder of husks and or fruit-stems with 95% ethanol to form an ethanol extract; concentrate the ethanol extract with decompression a to form the tannin extract. The second method for preparing the tannin extracts from the husks and or fruit-stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the husks and or fruit-stems with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extract the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins.

This invention provides a composition comprising the tannin extracts from the husks or fruit-stems of Wenguanguo. The extracts are comprised of tannins and others.

This invention provides two methods of producing a tannin extract from Wenguanguo leaves and its usage. The first method for preparing the tannin extract from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with 95% ethanol to form an ethanol extract; concentrate the ethanol extract with decompression a to form the tannin extract.

The second method for preparing the tannin extract from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extract the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract containing tannins.

This invention provides a composition comprising the tannin extract from the leaves of Wenguanguo. The extract comprises tannins and others.

This invention provides two methods of producing tannin extract from Wenguanguo branches and stems and its usage. The first method for preparing the extracts comprising tannins from branches or stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of branches or stems with 95% ethanol to form an ethanol extract; concentrate the ethanol extract with decompression a to form the tannin extract.

The second method for preparing the tannin extract from the branches or stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the branches or stems and with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extract the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins. This invention provides a composition comprising the tannin extract from the branch or stem of Wenguanguo. The extract comprises tannins and others.

This invention provides two methods of producing tannin extract from Wenguanguo kernels and its usage. The first method for preparing the tannin extract from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with 95% ethanol to form an ethanol extract; concentrating the ethanol extract with decompression to form the extract comprising tannins.

The second method for preparing the extracts containing tannins from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extracting the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract containing tannins.

This invention provides a composition comprising the tannin extract from kernels of Wenguanguo. The extract comprises tannins and others.

This invention provides two methods of producing tannin extract from Wenguanguo roots and its usage. The first method for preparing the tannin extract from the roots of Wenguanguo comprises the following steps: extracting Wenguanguo powder of roots with 95% ethanol to form an ethanol extract; concentrating the ethanol extract with decompression to form the tannin extract. The method-2 for preparing the tannin extract from the root of Wenguanguo comprises the following steps: extracting Wenguanguo powder of the root with a solvent of acetone-water at a ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extracting the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins.

This invention provides a composition comprising the tannin extracts from the roots of Wenguanguo. The extracts comprise tannins and others.

This invention provides two methods of producing tannin extract from Wenguanguo barks and its usage. The method-1 for preparing the tannin extract from the barks of Wenguanguo comprises the following steps: extracting Wenguanguo powder of barks with 95% ethanol to form an ethanol extract; concentrating the ethanol extract with decompression to form the tannin extract. The second method for preparing the tannin extract from the barks of Wenguanguo comprising the following steps: extracting Wenguanguo powder of the barks with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extracting the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins.

This invention provides a composition comprising the tannin extracts from the barks of Wenguanguo. The extracts comprise tannins and others.

This invention provides a method for preventing cerebral aging, improving memory, improving cerebral functions and curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer's disease, autism, brain trauma, Parkinson's disease and other diseases caused by cerebral dysfunctions, and treating arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder and treating impotence and premature ejaculation.

According to the theory of traditional Chinese medicine, enuresis, frequent micturition and urinary incontinence are caused by “deficiency in kidney (“shen”)”. Therefore, they are treated by using Chinese herbs which can tone the kidney, such as Ginseng Bajitian, Roucongrong Duzhong and Cordyceps. These tonifying herbs can strengthen function of the kidney and regulate water metabolism of human's body through the “kidney pathway” that will help with curing the enuresis, frequent micturition and urinary incontinence.

The Wenguanguo extracts of the present invention can also be used to treat the enuresis, frequent micturition and urinary incontinence. However, the Wenguanguo extracts cure the enuresis, frequent micturition and urinary incontinence through the “bladder pathway” to regulate water metabolism of human's body and urination. The Wenguanguo extracts of the present invention stimulate the growth of the bladder. See FIG. 10A. The Wenguanguo extracts of the present invention increase the capacity of bladder and function of bladder controlling the urination. See Experiment 15 and Experiment 15A. In another aspect of the present invention, Wenguanguo extracts, when used with the “kidney pathway” herbs to treat the enuresis, frequent micturition and urinary incontinence, will strengthen both the pathways of kidney and bladder, and then will produce better treatment results.

This invention provides the medicines or health foods which further comprise Vitamin B, Vitamin D, Vitamin K, grape seed extract and other antioxidants, Cordyceps or its extract, gingko or its extract, Panax ginseng and P. quinquefolium or their extracts, Huangpi (Clausena lansium) or its extracts, Echinacea or its extract, St John's Wort (Hypericum perforatum) or its extract, Gegen (Pueraria lobata) or its extract, Tianma (Gastrodia elata) or its extract, Armillariella mellea or its extract, Danshen (Salvia miltiorrhiza) or its extract, Sanqi (Panax notoginsen) or its extract, Monascus or Honqu (Red yeast rice), Huanqi (Hedysarum polybotrys) or its extract, Dihuang (Rehmannia glutinosa) or its extract, Danggui (Angelica sinensis), Yuanzhi (Polygala tenuifoila) or its extract, Lingzhi (Ganoderma spp.) or its extracts, Fuling (Poria cocos) or its extract, enokitake (Flammulina velutipes) or its extract, Gan Cao (Glycyrrhiza uralensis Fisch) or its extract, Huperzine A, Lacithin, Metrifonate, Nocetile, folic acid, amino acids, creatine, fiber supplement, or any combination thereof.

This invention provides a process of producing a coumarin extract from the kernels of Wenguanguo and their applications. The methods for preparing the coumarin extracts from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and drying them to form the kernel powder; extracting the kernel powder with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form an ether extract; neutralizing the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extracts from the kernels of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing a coumarin extract from the roots of Wenguanguo and their applications. The methods for preparing the coumarin extract from roots of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the root with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extracts from the roots of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing a coumarin extract from the barks of Wenguanguo and their applications. The methods for preparing the coumarin extract from barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the bark with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extract from the barks of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing an aqueous extract from the husks or fruit-stems of Wenguanguo and their applications. The method for preparing the water extracts from the husks or fruit-stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the husk or fruit-stem with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from the husks or fruit-stems of Wenguanguo The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the leaves of Wenguanguo and their applications. The method for preparing the water extracts from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from leaves of Wenguanguo. The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the branches or stems of Wenguanguo and their applications. The method for preparing the water extracts from branches or stems of Wenguanguo comprise the following steps: extracting the Wenguanguo powder of the branches or stems with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrating the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from the branches or stems of Wenguanguo. The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the kernels of Wenguanguo and their applications. The method for preparing the water extracts from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from kernels of Wenguanguo. The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the roots of Wenguanguo and their applications. The method for preparing the water extracts from the roots of Wenguanguo comprises the following steps: extracting Wenguanguo powder of the roots with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrating the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from the roots of Wenguanguo The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the barks of Wenguanguo and their applications. The method for preparing the water extracts from the barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the barks with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extracts from the barks of Wenguanguo The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an alkaloid extract from the husks of Wenguanguo and their applications. The methods for preparing the alkaloid extracts from the husks and fruit-stems of Wenguanguo comprising the following steps: extracting Wenguanguo powder of the husks or fruit-stems with water at a ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extract the alkalified aqueous extract by toluol to form a toluol extract; the toluol extract flows through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression a to form crude alkaloids.

This invention provides a composition comprising the alkaloid extract from the husks or fruit-stems of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the leaves of Wenguanguo and their applications. The methods for preparing the alkaloid extract from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with water at a ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collecting and alkalifying the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extracting the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression to form the alkaloid extract.

This invention provides a composition comprising the alkaloid extract from the leaves of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the branches and stems of Wenguanguo and their applications. The methods for preparing the extracts containing alkaloids from branches or stems of Wenguanguo comprising the following steps: extracting Wenguanguo powder of the branches or stems with water at ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extracting the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression to form the alkaloid extract.

This invention provides a composition comprising the extract containing crude alkaloids from the branches or stems of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the kernels of Wenguanguo and their applications. The methods for preparing the alkaloid extract from kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grounding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with water at ratio of 1:6 for 3-4 times, 10-15 hours for each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extract the alkalified aqueous extract by toluol to form a toluol extract; the toluol extract flows through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression to form the alkaloid extract.

This invention provides a composition comprising the alkaloid extract from the kernels of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the roots of Wenguanguo and their applications. The methods for preparing the alkaloid extract from the roots of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the Wenguanguo roots with water at a ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collecting and alkalifying the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extracting the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression a to form crude alkaloids.

This invention provides a composition comprising the alkaloid extract from the roots of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the barks of Wenguanguo and their applications. The methods for preparing the alkaloid extract from the barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the barks with water at ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extract the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression a to form crude alkaloids.

This invention provides a composition comprising the alkaloid extract from the barks of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing extract containing organic acids from husks and fruit-stems and their applications. The methods for preparing the extracts containing organic acids from the husks or fruit-stems of Wenguanguo comprise the following steps: extract Wenguanguo powder of the husks and or fruit-stems with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO₃ solution to form a NaHCO₃ extract; acidize and filter the NaHCO₃ extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising crude organic acids from the husks of Wenguanguo. The extract comprising aromatic organic acids, fatty organic acids, terpenoid organic acids and others

This invention provides a process of producing extract contains organic acids from leaf and their applications. The methods for preparing the extracts containing organic acids from the leaves of Wenguanguo comprise the following steps: extract Wenguanguo powder of the leaves with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO₃ solution to form a NaHCO₃ extract; acidize and filter the NaHCO₃ extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising the extract comprising crude organic acids extract from the leaves of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract contains organic acids from branches and stems and their applications. The methods for preparing the extracts comprising organic acids from the branches or stems of Wenguanguo comprise the following steps: extract Wenguanguo powder of the branches or stems with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO₃ solution to form a NaHCO₃ extract; acidize and filter the NaHCO₃ extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising the crude organic acids extract from the branches and stems of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract comprise organic acids from kernels and their applications. The methods for preparing the extracts comprising organic acids from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grounding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO₃ solution to form a NaHCO₃ extract; acidize and filter the NaHCO₃ extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising crude organic acids extract from the kernels of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract contains organic acids from the roots of Wenguanguo and their applications. The methods for preparing the extracts containing organic acids from the roots of Wenguanguo comprise the following steps: extract Wenguanguo powder of the roots with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO₃ solution to form a NaHCO₃ extract; acidize and filter the NaHCO₃ extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising the extract comprising crude organic acids from the roots of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract comprising organic acids from barks of Wenguanguo and their applications. The methods for preparing the extracts containing organic acids from the barks of Wenguanguo comprise the following steps: extract Wenguanguo powder of the bark with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO₃ solution to form a NaHCO₃ extract; acidize and filter the NaHCO₃ extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising the extract comprising crude organic acids from the barks of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides two methods of producing a tannin extract from Wenguanguo husks and fruit-stems and its usage. The first method for preparing the tannin extract from the husks or fruit-stems of Wenguanguo comprises the following steps: extracting Wenguanguo powder of husks and or fruit-stems with 95% ethanol to form an ethanol extract; concentrate the ethanol extract with decompression a to form the tannin extract. The second method for preparing the tannin extracts from the husks and or fruit-stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the husks and or fruit-stems with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extract the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins.

This invention provides a composition comprising the tannin extracts from the husks or fruit-stems of Wenguanguo. The extracts are comprised of tannins and others.

This invention provides two methods of producing a tannin extract from Wenguanguo leaves and its usage. The first method for preparing the tannin extract from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with 95% ethanol to form an ethanol extract; concentrate the ethanol extract with decompression a to form the tannin extract.

The second method for preparing the tannin extract from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extract the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract containing tannins.

This invention provides a composition comprising the tannin extract from the leaves of Wenguanguo. The extract comprises tannins and others.

This invention provides two methods of producing tannin extract from Wenguanguo branches and stems and its usage. The first method for preparing the extracts comprising tannins from branches or stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of branches or stems with 95% ethanol to form an ethanol extract; concentrate the ethanol extract with decompression a to form the tannin extract.

The second method for preparing the tannin extract from the branches or stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the branches or stems and with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extract the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins. This invention provides a composition comprising the tannin extract from the branch or stem of Wenguanguo. The extract comprises tannins and others.

This invention provides two methods of producing tannin extract from Wenguanguo kernels and its usage. The first method for preparing the tannin extract from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with 95% ethanol to form an ethanol extract; concentrating the ethanol extract with decompression to form the extract comprising tannins.

The second method for preparing the extracts containing tannins from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extracting the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract containing tannins.

This invention provides a composition comprising the tannin extract from kernels of Wenguanguo. The extract comprises tannins and others.

This invention provides two methods of producing tannin extract from Wenguanguo roots and its usage. The first method for preparing the tannin extract from the roots of Wenguanguo comprises the following steps: extracting Wenguanguo powder of roots with 95% ethanol to form an ethanol extract; concentrating the ethanol extract with decompression to form the tannin extract. The method-2 for preparing the tannin extract from the root of Wenguanguo comprises the following steps: extracting Wenguanguo powder of the root with a solvent of acetone-water at a ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extracting the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins.

This invention provides a composition comprising the tannin extracts from the roots of Wenguanguo. The extracts comprise tannins and others.

This invention provides two methods of producing tannin extract from Wenguanguo barks and its usage. The method-1 for preparing the tannin extract from the barks of Wenguanguo comprises the following steps: extracting Wenguanguo powder of barks with 95% ethanol to form an ethanol extract; concentrating the ethanol extract with decompression to form the tannin extract. The second method for preparing the tannin extract from the barks of Wenguanguo comprising the following steps: extracting Wenguanguo powder of the barks with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extracting the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins.

This invention provides a composition comprising the tannin extracts from the barks of Wenguanguo. The extracts comprise tannins and others.

This invention provides a method for preventing cerebral aging, improving memory, improving cerebral functions and curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer's disease, autism, brain trauma, Parkinson's disease and other diseases caused by cerebral dysfunctions, and treating arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder and treating impotence and premature ejaculation.

According to the theory of traditional Chinese medicine, enuresis, frequent micturition and urinary incontinence are caused by “deficiency in kidney (“shen”)”. Therefore, they are treated by using Chinese herbs which can tone the kidney, such as Ginseng Bajitian, Roucongrong Duzhong and Cordyceps. These tonifying herbs can strengthen function of the kidney and regulate water metabolism of human's body through the “kidney pathway” that will help with curing the enuresis, frequent micturition and urinary incontinence.

The Wenguanguo extracts of the present invention can also be used to treat the enuresis, frequent micturition and urinary incontinence. However, the Wenguanguo extracts cure the enuresis, frequent micturition and urinary incontinence through the “bladder pathway” to regulate water metabolism of human's body and urination. The Wenguanguo extracts of the present invention stimulate the growth of the bladder. See FIG. 10A. The Wenguanguo extracts of the present invention increase the capacity of bladder and function of bladder controlling the urination. See Experiment 15 and Experiment 15A. In another aspect of the present invention, Wenguanguo extracts, when used with the “kidney pathway” herbs to treat the enuresis, frequent micturition and urinary incontinence, will strengthen both the pathways of kidney and bladder, and then will produce better treatment results.

This invention provides the medicines or health foods which further comprise Vitamin B, Vitamin D, Vitamin K, grape seed extract and other antioxidants, Cordyceps or its extract, gingko or its extract, Panax ginseng and P. quinquefolium or their extracts, Huangpi (Clausena lansium) or its extracts, Echinacea or its extract, St John's Wort (Hypericum perforatum) or its extract, Gegen (Pueraria lobata) or its extract, Tianma (Gastrodia elata) or its extract, Armillariella mellea or its extract, Danshen (Salvia miltiorrhiza) or its extract, Sanqi (Panax notoginsen) or its extract, Monascus or Honqu (Red yeast rice), Huanqi (Hedysarum polybotrys) or its extract, Dihuang (Rehmannia glutinosa) or its extract, Danggui (Angelica sinensis), Yuanzhi (Polygala tenuifoila) or its extract, Lingzhi (Ganoderma spp.) or its extracts, Fuling (Poria cocos) or its extract, enokitake (Flammulina velutipes) or its extract, Gan Cao (Glycyrrhiza uralensis Fisch) or its extract, Huperzine A, Lacithin, Metrifonate, Nocetile, folic acid, amino acids, creatine, fiber supplement, or any combination thereof.

There are many different periods of sleep a person goes through. These include Slow-Wave-Sleep 1 (SWS 1), Slow-Wave-Sleep 2 (SWS 2), Slow-Wave-Sleep 3 (SWS 3) Slow-Wave-Sleep 4 (SWS 4) and Rapid Eye Movement (REM). SWS 1 and SWS 2 are both periods of light sleep where it is relatively easy to wake someone up. Light sleep is usually more frequent in the second half of sleep. SWS 3 and SWS 4 are both periods of deep sleep, where it is difficult to wake the sleeper. Deep sleep is more frequent in the first half of sleep and each period will get shorter each time afterward. REM is a period of sleep in which people have their most vivid dreams. The wave patterns are similar to the patterns in which a person is awake. However, it is difficult to wake someone up who is in this state of sleep. The sleep cycle of a typical person can be described as follows:

SWS1, SWS2, SWS3, SWS4, SWS3, SWS2, REM, SWS1, SWS2, SWS3, SWS4, SWS3, SWS2, REM. (See FIG. 62)

However, the above sequence may not be in a fixed order. The sleep status may shift from SWS 4 to SWS1 or wake up because the body turns from one side to the other. It may shift to the SWS2 status after the movement. The interval between two REM is about 90 min. For healthy people, SWS1 will occupy about 5% of sleep, SWS2 will occupy about 50% of sleep, SWS3 will occupy about 10% of sleep, SWS4 will occupy about 10% of sleep and REM will occupy about 25% of sleep. Since a person in SWS1 and SWS2 can easily be woken, a healthy person has enough opportunities to wake up to urinate. However if a person's sleeping status is mostly in SWS4, he has less chance of waking up when the bladder is full. It is difficult for him to break through the barriers of deep sleep. Then enuresis occurs. This invention relates to a plant extract, including Wenguanguo, for preventing enuresis.

This invention provides the extract of Wenguanguo for inhibiting the uptake of 5-hydroxytryptamine (5HT) in a subject.

5-HT controls and modulates a sleep factor that sustains and increases deep sleep. Inhibiting the uptake of 5HT will decrease deep sleep. People who spend too much time in SWS 3 and SWS 4 are unable to awaken from their sleep when their bladder is full because their sleep is too deep. This is the reason that enuresis often occurs during SWS 3 and SWS 4.

This invention provides the extract of Wenguanguo for increasing the activity of Dopamine in a subject thereby making the central nerve system of said subject alert.

This invention provides the extract of Wenguanguo for increasing the secretion of antidiuretic hormone (ADH) in a subject, which reduces urine in said subject.

This invention provides the extract of Wenguanguo for modulating the release, breakdown and uptake of Acetylcholine (Ach) and its receptors in a subject. The said extracts of this invention inhibits the deep sleep created by 5HT and increase REM sleep.

This invention provides the extract of Wenguanguo for preventing sleep paralysis in a subject.

This invention provides the extract of Wenguanguo for providing alertness to a sleeping subject.

This invention provides the extract for helping the growth of the bladder and sphincter.

An immature bladder and sphincter cannot control the process and action of urination. By accelerating the growth of the bladder and the sphincter, this problem will be overcome, and enuresis will not occur.

This invention provides the extract of Wenguanguo against cancer growth. The cancer includes, but is not limited to bladder cancer, cervix cancer, prostate cancer, lung cancer, breast cancer, leukocytes cancer, colon cancer, liver cancer, bone cancer, skin cancer, brain cancer, and ovary cancer

This invention provides the extract of Wenguanguo inhibit tumor activities.

This invention provides the pathways interacted by compounds isolated from Xanthoceras Sorbifolia. In an embodiment, a compound has the formula C₅₇H₈₈O₂₃ and the chemical name 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β, 15α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene, designated herein as “Structure Y”, and derivative compounds which are effective against cancer. In another embodiment, the compounds of the present invention comprise the chemical structures designated herein as “Structure Y1”, “Structure R1”, “Structure 1 to 4”, “Structure Y-a to Y-c” and “Structure Y1-a to Y1-c”, “Structure Y1-1 to Y1-4” and their derivatives. See FIG. 31-40.

They regulate the receptors or components of cells. The compounds can be isolated from the plant called Xanthoceras Sorbifolia or can be synthesized.

The compounds of the present invention have structures as shown below: Structure Y (also shown in FIG. 1)

3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β, 15α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene

Structure 1 as shown in FIG. 31 Structure 2 as shown in FIG. 32 Structure 3 as shown in FIG. 33 Structure 4 as shown in FIG. 34 Structure Y-a as shown in FIG. 35 Structure Y-b as shown in FIG. 36 Structure Y-c as shown in FIG. 37 Structure Y1 (also shown in FIG. 2).

3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,16α, 21β, 22α, 28-pentahydroxyolean-12-ene

Structure Y1-a as shown in FIG. 38 Structure Y1-b as shown in FIG. 39 Structure Y1-c as shown in FIG. 40.

This invention further provides a compound comprising the following structure:

Structure R1: 3-O-[angeloyl-(1→3)-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β, 21β, 22α, 28-tetrahydroxyolean-12-ene

This invention further provides a compound comprising the following structure:

The structure of compound O54 is presented in the following figure.

The chemical name of compound-O54 is:

O54:3-O-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β, 21β, 22α, 28-tetrahydroxyolean-12-ene.

There are many components and pathways monitoring cell proliferation.

The Xanthoceras Sorbifolia compound or its derivatives work in the Wnt (Wingless-type MMTV integration site family member) signaling pathway. The Wnt signaling pathway is evolutionarily conserved and controls many events during the embryogenesis. This pathway regulates cell morphology, proliferation, motility and as well as cell apoptosis. It also plays an important role during tumorigenesis. The Wnt pathway has also been observed as inappropriately activated in several different types of cancers in humans.

In the nucleus, the target genes for Wnt signaling are normally kept silent by an inhibitory complex of gene regulatory proteins, e.g. the Groucho corepressor protein bound to the gene regulatory protein LEF-I/TCF. In the absence of a Wnt signal, some β-cartenin is bound to the cytosolic tail of cadherin proteins, and any cytosolic β-cartenin that becomes bound by the APC-axin-GSK-3β will trigger its ubiquitylation and degradation in proteasomes. The result is the decrease of intracellular amount of β-cartenin. However, when the Wnt binding to Frizzled (a seven transmembrane receptor) and LRP (Low density lipoprotein Receptor) activates Dishevelled (a cytoplasmic signaling protein) by a mechanism, this leads to the inactivation of GSK-β3 in the degradation complex by a mechanism which requires casein kinase I, as well as casein kinase II. The activity of the multiprotein complex of β-catenin-axin-adenomatous-polyposis coli (APC)-glycogen synthase kinase (GSK)-3β, which targets β-catenin by phosphorylation for degradation by the proteasome, is then inhibited by Dsh/Dvl (Dishevelled, dsh homolog 1). This then inhibits priming of β-catenin, and indirectly prevents the GSK-3β phosphorylation of β-catenin. When stimulated by Wnt, Dvl recruits the GSK-3 binding protein, GBP, to the multiprotein complex of β-catenin-axin-adenomatous-polyposis coli (APC)-glycogen synthase kinase (GSK)-3β. GBP then titrates GSK-β from axin, and in this way, phosphorylation of β-catenin is inhibited. Then, axin is sequestrated by LRP at the cell membrane. The result of all of this is an accumulation of cytosolic β-catenin. In the nucleus, β-catenin binds to LEF-I/TCF, displaces Groucho, and acts a co-activator to stimulate the transcription of Wnt target genes.

Xanthoceras Sorbifolia compositions regulate the components related to Wnt pathways or its receptors, thereby stopping the proliferation of cancer cells.

The compound or its derivatives work in the Mitogens, Ras and a MAP (Mitogen activation protein) kinase pathway. Mitogens stimulate cell division. The binding of mitogens to cell-surface receptors leads to the activation of Ras and a MAP kinase cascade. One effect of this pathway is the increased production of the gene regulatory protein Myc. Myc increases the transcription of several genes, including the gene encoding cyclin D and a subunit of the SCF ubiquitin ligase. The resulting increase in G₁-Cdk and G₁/S-Cdk activities promotes Rb phosphyorylation and activation of the gene regulatory protein E2F, resulting in S-phase entry, in which G₁-Cdk activity initiates Rb phosphorylation, in turn inactivating Rb and freeing E2F to activate the transcription of S-phase genes including the genes for a G₁/S-cyclin (cyclin E) and S-cyclin (cyclin A). The resulting appearance of G₁/S-Cdk and S-Cdk further enhances Rb phosphorylation, forming a positive feedback loop, and the E2F acts back to stimulate the transcription of its own gene, forming another positive feedback loop. Myc may also promote E2F activity directly by stimulating the transcription of the E2F gene. The result is the increased transcription of genes entry into S phase. However if this pathway is overactive, it will cause cancer cell growth.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the Ras-MAP kinase cascade so that the pathway is not overactive.

The compound or its derivatives work in Ras-dependent or Myc pathway. Sometimes the mutation of amino acid in Ras causes the protein to become permanently overactive, stimulating the Ras-dependent signal pathways overactive in absence of mitogenic stimulation. Similarly, mutations that cause an overexpression of Myc promote excessive cell growth, which in turn promotes the development of cancer.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components of the Ras-dependent or Myc pathway to make sure it is not overactive.

The compound or its derivatives reactivate the abnormal cell checkpoint mechanism. Inside the cell, there is a checkpoint mechanism which detects abnormal mitogenic stimulation and causes abnormally overactive cells to go into apoptosis. However this mechanism is not active in cancer cells due to mutations in the genes that encode essential components of the checkpoint responses. If the mutation happens in the checkpoint mechanism, the cancer cell will grow and divide endlessly.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia reactivate the checkpoint mechanism to stop the cancer cell growth.

The compound or its derivatives affect the extracellular growth signaling pathways. The extracellular growth factors that stimulate cell growth are bound to receptors on the cell surface and activate intracellular signaling pathways. It activates the enzyme PI3-kinase, which promotes protein synthesis, at least partly through the activation of EIF4e and phosphorylated S6 kinase, resulting in increased mRNA translation and then a stimulation of cell growth.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components or receptor relate to extracellular growth. It binds the receptor of ovarian cancer cells so as to stop the cancer cell growth.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components relating to Ras and MAP Kinase, which ceases ovarian cancer cell growth.

The compound or its derivatives affect the intracellular mechanism. Cell division is also controlled by an intracellular mechanism that can limit cell proliferation. In normal cells, the Myc protein acts in the nucleus as a signal for cell proliferation. Large quantities of Myc can cause the cell to proliferate in excess and form a tumor.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components or receptor of the Myc cell's proliferation to stop the tumor cells from dividing.

The compound or its derivatives affect the TGF-alpha signaling pathway. TGF-alpha is produced by keratincytes, macrophages, hepatocytes, and platelets. Its synthesis is stimulated by the infection by viruses. TGF-Alpha induces the long term proliferation of murine and chicken immature hematopoietic progenitor cell such as BFU-E without causing differentiation. It also induces the terminal differentiation of BFU-Ecell into erythrocytes. TGF-Alpha stimulates the proliferation of cultured endothelial cells. It plays an importance role in the vascularisation of tumor tissues.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components or receptor of TGF-alpha to suppress ovarian cancer and bladder cancer cell growth.

The compound or its derivative compounds affect the TGF-beta signaling pathway. TGF-beta regulates growth and proliferation of cells, blocking growth of many cell types. There are two TGF-beta receptors: Type 1 and Type 2. They are serine-threonine kinases that signal through the SMAD (Protein named after the first two identified, Sma in C. elegans and Mad in Drosophila) family of transcriptional regulators. The TGF-beta pathway and mutation in SMADs are associated with cancer in humans.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components or receptor of TGF-beta to suppress the ovarian cancer and bladder cancer cell growth.

The compound or its derivatives reactivate the cell functions which are damaged by DNA viruses. DNA tumor viruses cause cancer by interfering with cell cycle control Rb protein and the p53 protein. Mutation in p53 gene will allow cancer cells to survive and proliferate despite DNA damage. The papillomanius uses the proteins E6 and E7 to release the p53 and Rb respectively. This action activates mutated cells, allowing them to survive and then divide and accumulate. The accumulation of damaged cells can lead to cancer.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the proteins E6 and E7 and release the proteins Rb and p53, which will prevent abnormal cells from dividing. It also regulates or reacts with the protein, causing the cancer cells to die.

The compound or its derivatives affect the p53 signaling pathway. p53 helps multi-cellular organisms cope safely with DNA damage and other stressful cellular events, stopping cell proliferation in circumstances where it would be dangerous. Cancer cells tend to contain large quantities of mutant p53 protein, suggesting that the genetic accidents they undergo or the stresses of growth in an inappropriate environment created the signals that normally activate the p53 protein. Thus, the loss of p53 activity can be extremely dangerous in relation to cancer because it allows mutant cells to continue through the cell cycle. It also allows them to escape apoptosis. So, if their DNA is damaged, some cells will die but the cells which survive will carry on dividing without pausing to repair the damage. This may cause the cells to die, or they could survive and proliferate with a corrupted genome, which could lead to loss of both tumor suppressor genes and the activation of oncogenes, for example by gene amplification. Gene amplification could enable cells to develop resistance against therapeutic drugs.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components and receptor of the p53 pathway, which stops the cancer cells from dividing.

The compound or its derivatives affect the cell suicide signaling pathway. All cells with a nucleus contain various inactive procaspases, awaiting a signal before destroying the cell. Each suicide protease is made as an inactive proenzyme called procaspase. It is usually activated by proteolytic cleavage by another member of the caspase family. Two of the cleaved fragments come together to form the active part of the caspase, and the active enzyme is thought to be a tetramer of two of these two parts. Each activated caspase molecule can cleave many procaspase molecules, which in turn activates more molecules. Through a chain reaction or cascade, this leads to the explosive action of a large number of procaspase molecules. Then, some of the activated procaspases cleave a number of key proteins in the cell, including specific cytosolic proteins and nuclear-lamins leading to the controlled death of the cell.

Activating the death receptor on the outside of the cell can also trigger inactive procaspases. For example, killer lymphocytes can cause apoptosis by producing the protein Fas on the surface of the targeted cell. These clusters of Fas protein then recruit intracellular adaptor proteins that bind and aggregate procaspase-8 molecules. These then cleave and activate one another. The activated caspase-8 molecules then activate downstream procaspases to induce apoptosis.

However in cancer cells, the signal to destroy the cell is blocked, due to gene mutation. This means that the cancer cells continue to divide, thereby causing a tumor.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia unblock the suicide signals, allowing cancer cells to destroy themselves. Structure showed in FIG. 67.

This invention provides a method for inhibiting tumor cell growth comprising contacting an amount of the above-described compound, wherein R1, R2, R3, R4 are short aliphatic chain and R5 contains an oxyl group; and a pharmaceutically acceptable carrier effective to inhibit growth of said tumor cells.

This invention provides a method for inhibiting tumor cell growth comprising contacting an amount of the above-described compounds.

This invention provides a method for inhibiting tumor cell growth comprising contacting an amount of the compound comprising: a sugar; a triterpene or Sapogenin; side chain at Carbon 21 and 22 or Angeloyl groups, operatively linked form the compound; and a pharmaceutically acceptable carrier.

Structure showed in FIG. 67.

This invention provides a method for inhibiting tumor cell growth in a subject comprising administering to the above-described subject, wherein R1, R2, R3, R4 are short aliphatic chain and R5 contains an oxyl group; effective to inhibit growth of said tumor cells and a pharmaceutically acceptable carrier.

This invention provides a method of for inhibiting tumor cell wherein R1=R2=R3=R4=CH3 and R5 contains an oxyl bond.

This invention provides a method for inhibiting tumor cell growth comprising contacting an amount of the above-described compounds.

This invention provides a compound consist of a triterpene or sapongenin, sugar moiety connected to the backbone. A sugar was linked the C21 position where two angeloyl groups were attached. This compound has the anti-cancer activity

This invention provides a method for inhibiting tumor cell growth comprising contacting an amount of the compound is a triterpene or sapongenin with any two of angeloyl group or tigloyl group or senecioyl group or their combinations attach to carbon 21 and 22, or any two of angloyl group or tigloyl group or senecioyl group or their combinations attached to a sugar moiety which bonds to carbon 21 or 22.

The structures of this invention or its derivative can be synthesis or from biological sources.

This invention will be better understood from the examples which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.

EXPERIMENTAL DETAILS Experiment 1 Herb Extraction

(a) extracting Xanthoceras Sorbifolia powder of husks or branches or stems or leaves or kernels or roots or barks with organic solvent at ratio of 1:2 for 4-5 times for 20-35 hours for each time to form an organic extract; (b) collecting the organic extract; (c) refluxing the organic extract for 2-3 times at 80° C. to form second extracts; (d) removing the organic solvent from the second extract; and (e) Drying and sterilizing the extract to form a Xanthoceras Sorbifolia extract powder.

Experiment 2 Analysis of Xanthoceras Sorbifolia Extract Components by HPLC Chromatography Methods

HPLC. A C-18 reverse phase μbondapak column (Water P/N 27324) was equilibrated with 10% acetonitrile, 0.005% Trifluoroacetic acid (equilibration solution). An extract of Xanthoceras Sorbifolia prepared using the methods of the present invention was dissolved in equilibration solution (1 mg/ml) before being applied onto the column. 20 ug of samples was applied into column. Elution conditions: Fractions were eluted (flow rate 0.5 ml/min.) with acetonitrile (concentration gradient from 10% to 80% in 70 min) and then remains at 80% for 10 min (70-80 min). The acetonitrile concentration then decreased to 10% (80-85 min) and remained at 10% for 25 min (85-110 min). The fractions were monitored at 207 nm and recorded in chart with a chart speed of 0.25 cm/min and with a OD full scale of 0.128.

Instruments. Waters Model 510 Solvent Delivery System; Waters 484 tunable Absorbance Detector; Waters 745/745B Data Module

Absorbance analysis. The absorption profile of Xanthoceras Sorbifolia extract at various wavelengths was determined. An extract of Xanthoceras Sorbifolia of the present invention was dissolved in 10% acetonitrile/TFA and scanned at 200-700 nm with a spectrophotometer [Spectronic Ins. Model Gene Sys2].

Results

HPLC. About 60-70 peaks can be accounted for in the profile. Among them four are major peaks, 10 are medium size and the rest are small fractions. The major peaks are labelled with a to z following increased concentration of acetonitrile elution. See FIG. 6.

Absorption maximum. Three absorption maximum were identified for Xanthoceras Sorbifolia plant extract; 207 nm, 278 nm and 500 nm. See FIG. 41.

Experiment 3

Screening of Cytotoxicity of Xanthoceras Sorbifolia Extract with Cancer Cells Derived from Different Human Organs Using MTT Assay

Methods and Materials

Cells. Human cancer cell lines were obtained from American Type Culture Collection: HTB-9 (bladder), HeLa-S3 (cervix), DU145 (prostate), H460 (lung), MCF-7 (breast), K562 (leukocytes), HCT116 (colon), HepG2 (liver), U2OS (bone), T98G (brain) and OVCAR-3 (ovary). Cells were grown in culture medium (HeLa-S3, DU145, MCF-7, Hep-G2 and T98G in MEN (Earle's salts); HTB-9, H460, K562, OVCAR-3 in RPMI-1640; HCT-116, U2OS in McCoy-5A) supplemented with 10% fetal calf serum, glutamine and antibiotics in a 5% CO2 humidified incubator at 37° C.

MTT assay. The procedure for MTT assay followed the method described in (Carmichael et al., 1987) with only minor modifications. Cells were seeded into a 96-wells plate at concentrations of 10,000/well (HTB-9, HeLa, H460, HCT116, T98G, OVCAR-3), 15,000/well (DU145, MCF-7, HepG2, U2OS), or 40,000/well (K562), for 24 hours before drug-treatment. Cells were then exposed to drugs for 48 hours (72 hours for HepG2, U2OS, and 96 hours for MCF-7). After the drug-treatment, MTT (0.5 mg/ml) was added to cultures for an hour. The formation of formazan (product of the reduction of tetrazolium by viable cells) was dissolved with DMSO and the O.D. at 490 nm was measured by an ELISA reader [Dynatech. Model MR700]. The MTT level of cells before drug-treatment was also measured (T0). The % cell-growth (% G) is calculated as:

% G=(TD−T0/TC−T0)×100  (1)

where TC or TD represent O.D. readings of control or drug-treated cells. When T0>TD, then the cytotoxicity (LC) expressed as % of the control is calculated as:

% LC=(TD−T0/T0)×100.

Results. Among the 10 cell lines studies, their sensitivity toward Xanthoceras Sorbifolia extract can be divided into four groups (most sensitive: Ovary. Sensitive: bladder, bone, prostate, and leukocyte, marginal sensitive: liver, breast, and brain; and lease sensitive: colon, cervix, and lung) (FIG. 8, 10A-D). Their IC50 values are listed in Table 3.1.

TABLE 3.1 IC50 values of Xanthoceras Sorbifolia Extract Determined in Different Cancer Cells IC50 determined by MTT assay Cancer cells from different organs (ug/ml) Ovary (most sensitive) 15-15 Bladder (sensitive) 45-50 Bone 40-55 Prostate 40-50 Leukocyte 45-50 Liver (marginal sensitive) 45-65 Breast 65 Brain 70-85 Colon (least sensitive) 90 Cervix 115 Lung 110

Xanthoceras Sorbifolia plant extract stimulate cell growth of bladder, bone and lung cells. See FIGS. 10A, 10D.

To invest Among these cell line studied, it was found that low concentrations of the igate the growth and inhibition components of the Xanthoceras Sorbifolia plant extract, the plant extract was fractionated. FIG. 5 shows the results of the screening of cell growth activity of fractions obtained after FPLC chromatography. The assay was conducted with bladder cells. The fractions obtained from FPLC as shown in FIG. 20 were used. As shown in this figure, that different components of Xanthoceras Sorbifolia extracts cause either growth or inhibition effects on cells. Only fraction 5962 (Fraction Y) causes cell inhibition. Fractions 610 and 1116 cause minor stimulation of cell growth. Abscissa: concentration (ug/ml). Ordinate: % Cell Growth (determined by MTT assay).

Experiment 4 Purification of Inhibition Components in the Xanthoceras Sorbifolia Extract

(A) Fractionation of Xanthoceras Sorbifolia Extracts Components with FPLC.

Methods

Column. Octadecyl functionalized silica gel; column dimension: 2 cm×28 cm; equilibrated with 10% acetonitrile-0.005% TFA.

-   -   Sample loading: 1-2 ml, concentration: 100 mg/ml in 10%         acetonitrile/TFA.     -   Gradient elution: 10-80% acetonitrile in a total volume of 500         ml.     -   Monitor absorption wavelength: at 254 nm.     -   Fraction Collector: 5 ml/fractions (collect from 10% to 72%         acetonitrile, total 90 fractions)     -   Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.

Results.

The elution profile shows 4-5 broad fractions. See FIG. 20. These fractions were analyzed with HPLC. Specific components, i.e., a-z as specified in FIG. 6, are then assigned in the FPLC fractions.

FPLC fractions are grouped into 7 pools and analyzed for cell growth activity with bladder cells with MTT assay. It was found only one pool (#5962) contains inhibition activity. See FIG. 5.

(B) Fractionation of Fraction #5962 with FPLC by a C18 Open Column with 64% Acetonitrile Isocratic Elution Methods

Column.

Octadecyl-functionalized silica gel; 50 ml; 2 cm×28 cm; equilibrated with 64% acetonitrile-0.005% TFA.

-   -   Sample loading: 0.2 ml, with concentration: 1-2 mg/ml in 65%         acetonitrile/TFA.     -   Elution: 64% acetonitrile isocratic.     -   Monitor absorption wavelength: at 254 nm.     -   Fraction Collector: 1 ml fraction (collect the first 90         fractions)     -   Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.

Results.

Fraction 5962 was further separated with an open ODS-C18 column using isocratic 64% acetonitrile elution. Two major fractions, i.e., X and Y, were collected. See FIG. 42. MTT assay showed that only the Y fraction has the inhibition activity. See FIG. 43.

(C) Analysis of Fraction Y with HPLC

Methods Column.

Waters μ-bondapak C18 (3.9 mm×300 cm).

-   -   Elution: 35% or 45% isocratic elution.     -   Flow rate: 0.5 ml/min; monitored at 207 nm with O.D. Scale of         0.128; chart speed: 0.25 cm/min.

Results.

On 45% isocratic analysis, Three fractions of Y were obtained (FIG. 44). (D) Final Isolation of Active Y Component with Preparative HPLC.

Methods

Column: A preparative HPLC column (Waters Delta Pak C18-300A);

-   -   Elution: 45% acetonitrile isocratic elution with flow rate of 1         ml/min.     -   Monitor at 207 nm;     -   Fractions (designated as Y1, Y2, etc.) were collected and         lyophilized.

Results.

Final separation of Y fractions was achieved by HPLC with a preparative column (FIG. 7). These fractions (compound Y1, Y2, Y3 and Y4) including the major fraction Y3 (designated as compound Y) were collected. Re-chromatography of the Compound Y showed a single peak in HPLC with a C18 reverse phase column. See FIG. 7A.

(E) Appearance and Solubility.

The pure Compound Y is amorphous white powder, soluble in aqueous alcohol (methanol, ethanol), 50% acetonitrile and 100% pyridine.

(F) Inhibition analysis of Compound Y with MTT assay.

Inhibition analysis of Compound Y was determined with MTT assay. The results indicated that (a) Compound Y has activity against ovarian cancer cells (OCAR-3) with IC50 value of 1.5 ug/ml which is 10-15 times more potent than the unpurified extract. See FIG. 3; and FIG. 8.

(b) Compound Y maintains its selectivity against ovarian cancer cells versus cervical cancer cells (HeLa). See FIG. 9.

Experiment 5

Determination of the Chemical Structure of Compound Y of Xanthoceras Sorbifolia Extract

Methods NMR Analysis.

The pure compound Y of Xanthoceras Sorbifolia were dissolved in pyridine-D5 with 0.05% v/v TMS. All NMR spectra were acquired using a Bruker Avance 600 MHz NMR spectrometer with a QXI probe (¹H/¹³C/¹⁵N/³¹P) at 298 K. The numbers of scans for 1D ¹H spectra were 16 to 128, depending on the sample concentration. 2D HMQC spectra were recorded with spectral widths of 6000×24,000 Hz and data points of 2024×256 for t₂ and t₁ dimensions, respectively. The numbers of scans were 4 to 128. 2D HMBC were acquired with spectral widths of 6000×30,000 Hz and data points of 2024×512 for t₂ and t₁ dimensions, respectively. The numbers of scans were 64. The 2D data were zero-filled in t₁ dimension to double the data points, multiplied by cosine-square-bell window functions in both t₁ and t₂ dimensions, and Fourier-transformed using software XWIN-NMR. The final real matrix sizes of these 2D spectra are 2048×256 and 2048×512 data points (F2×F1) for HMQC and HMBC, respectively.

Mass Spectral Analysis.

The mass of samples was analyzed by (A) MALDI-TOF Mass Spectrometry and by (B) ESI-MS Mass spectrometry. (A) Samples for MALDI-TOF were first dissolved in acetonitrile, then mixed with the matrix CHCA (Alpha-cyano-4-hydroxycinnamic acid, 10 mg CHCA/mL in 50:50 water/acetonitrile and 0.1% TFA in final concentration). The molecular weight was determined by the high resolution mass spectroscope analysis with standards. (B) For ESI, the sample was analyzed with LCQ DECA XP Plus machine made by Thermo Finnigan. It is ionized with ESI source and the solvent for the compound is acetonitrile.

Results.

The profile of the proton NMR is presented in FIG. 11. The 2D NMR profiles of HMQC and HMBC are shown in FIGS. 12 and 13, respectively.

Table 5.1 summarizes the 2D NMR chemical shift data and shows the assignment of functional groups derived from these chemical shifts. Based on these data and analysis, the structure of compound Y is assigned and shown below.

Structure of Compound Y:

The chemical name of compound Y is: 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β, 15α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene.

TABLE 5.1 ¹³C and ¹H NMR Data for Compound Y (in Pyridine-d₅)^(a) Position C H Key HMBC correlations  1 38.7 0.83, 1.40 C-3, C-5, C-9  2 26.4 1.81, 2.14 —  3 89.6 3.25, 1H, dd, 12.0/4.0 Hz C-23, C-24, GlcA C-1′  4 39.4 — —  5 55.3 0.78 —  6 18.5 1.55, 1.59 C-8, C-10  7 36.5 2.00, 2.10 C-5, C-9  8 41.2 — —  9 47.0 3.06 C-7, C-8, C-12, C-14, C-26 10 37.2 — — 11 23.7 1.74, 1.89 — 12 125.2 5.49, 1H, br s C-9, C-11, C-14, C-18 13 143.4 — — 14 47.5 — — 15 67.3 4.21 C-8, C-27 16 73.6 4.45 C-14, C-15, C-18 17 48.3 — — 18 40.8 3.07 C-12, C-13, C-14, C-16, C-19, C-20, C-28, 19 46.8 1.41, 1.69 — 20 36.2 — — 21 79.3 6.71, 1H, d, 10 Hz C-20, C-22, C-29, C-30, 21-O-Ang C-1′′′′ 22 73.5 6.32, 1H, d, 10 Hz C-16, C-17, C-21, C-28, 22-O-Ang C-1′′′′ 23 27.7 1.26, 3H, s C-3, C-4, C-5, C-24 24 16.5 1.16, 3H, s C-3, C-4, C-5, C-23 25 16.0 0.81, 3H, s C-1, C-5, C-9, C-10 26 17.3 0.99, 3H, s C-7, C-8, C-9, C-14 27 21.0 1.85, 3H, s C-8, C-13, C-14, C-15 28 62.9 3.50, 1H, d, 11.0 Hz, C-16, C-17, C-18, C-22 3.76, 1H, d, 11.0 Hz, 29 29.2 1.09, 3H, s C-19, C-20, C-21, C-30 30 20.0 1.32, 3H, s C-19, C-20, C-21, C-29 GlcA  1′ 104.9 4.89, 1H, d, 7.8 Hz C-3  2′ 79.1 4.38 GlcA C-1′, C-3′, Gal C-1″  3′ 86.1 4.20 GlcA C-2′, C-4′, Ara C-1′′′  4′ 71.5 4.42 GlcA C-3′, C-5′, C-6′  5′ 78.0 4.52 GlcA C-4′, C-6′  6′ 171.9 — — Gal  1″ 104.6 5.32, 1H, d, 7.7 Hz GlcA C-2′  2″ 73.6 4.42 Gal C-1″, C-3″  3″ 74.9 4.10 Gal C-2″  4″ 69.5 4.56 Gal C-2″, C-3″  5″ 76.4 3.94 Gal C-4″, C-6″  6″ 61.6 4.43, 4.52 Gal C-4″, C-5″ Ara-f  1′′′ 110.6 6.03. 1H, br s GlcA C-3′, Ara C-2′′′, C-4′′′  2′′′ 83.4 4.94 Ara C-3′′′  3′′′ 78.3 4.78 Ara C-2′′′  4′′′ 85.2 4.82 Ara C-5′′′  5′′′ 62.2 4.12, 4.28 Ara C-3′′′ 21-O-Ang  1′′′′ 167.7 — —  2′′′′ 129.6 — —  3′′′′ 137.2 5.96, 1H, dq, 7.0/1.5 Hz Ang C-1′′′′, C-4′′′′, C-5′′′′  4′′′′ 15.5 2.10, 3H, dq, 7.0/1.5 Hz Ang C-2′′′′, C-3′′′′  5′′′′ 20.8 2.00, 3H, s Ang C-1′′′′, C-2′′′′, C-3′′′′ 22-O-Ang  1′′′′ 167.9 — —  2′′′′ 129.8 — —  3′′′′ 136.3 5.78, 1H, dq, 7.0/1.5 Hz Ang C-1′′′′, C-4′′′′, C-5′′′′  4′′′′ 15.5 1.93, 3H, dq, 7.0/1.5 Hz Ang C-2′′′′, C-3′′′′  5′′′′ 20.5 1.74, 3H, s Ang C-1′′′′, C-2′′′′, C-3′′′′ ^(a)The data were assigned based on HMQC and HMBC correlations.

FIGS. 14 and 15 show the mass spectrum of Compound Y as determined by MALDI-TOF and ESI-MS techniques. Based on these data, the mass of compound Y is 1140.57 which agrees with the theoretical mass of the compound Y.

Conclusion

The active compound Y isolated from extract of Xanthoceras Sorbifolia is a triterpenoid saponins with three sugars and biangeloyl groups attached to the C21 and C22 positions of the backbone. The formula of Y is C₅₇H₈₈O₂₃, and the chemical name of Compound Y is: 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β, 15α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene.

Experiment 6 Determination of the Chemical Structure of Compound Y1 of Xanthoceras Sorbifolia Extract Methods

The method for NMR and MS analysis for compound Y1 are same as described in Experiment 5.

Results.

The profile of the H-NMR of Y1 is presented in FIG. 16. The 2D NMR profiles of HMQC, HMBC and COSY are shown in FIGS. 17, 18 and 19, respectively.

Table 6.1 summarizes the 1D and 2D NMR chemical shift data and shows the assignment of functional groups derived from these chemical shifts.

TABLE 6.1 ¹³C and ¹H NMR Data for Compound Y1 (in Pyridine-d₅)^(a) Position C H  1 38.6 0.85, 1.33  2 26.3 1.86, 2.10  3 89.7 3.25, 1H, dd  4 39.5 —  5 55.5 0.75  6 18.3 1.40, 1.43  7 33.1 1.20, 1.50  8 40.0 —  9 46.7 1.69 10 36.5 — 11 22.5 2.30 12 123.6 5.36, 1H, br s 13 143.5 — 14 41.8 — 15 34.7 1.53, 1.73 16 68.5 4.45 17 48.2 — 18 39.9 3.04 19 47.6 1.30, 3.05 20 36.7 — 21 85.3 5.05, 1H, d 22 73.8 6.17, 1H, d 23 27.7 1.29, 3H, s 24 16.5 1.16, 3H, s 25 15.5 0.81, 3H, s 26 17.1 0.82, 3H, s 27 20.6 1.83, 3H, s 28 63.7 3.42, 1H, d, 3.60, 1H, d 29 29.9 1.42, 3H, s 30 19.9 1.37, 3H, s GlcA  1 105.0 4.88, 1H, d  2 79.0 4.37  3 86.0 4.20  4 71.6 4.43  5 78.0 4.50  6 171.8 — Gal  1 104.5 5.31, 1H, d  2 73.5 4.43  3 74.9 4.10  4 69.5 4.57  5 76.3 3.95  6 61.1 4.44, 4.53 Ara-f  1 110.9 6.04. 1H, br s  2 83.3 4.95  3 78.3 4.78  4 85.2 4.82  5 62.0 4.13, 4.31 21-O-Rha  1 105.1 4.92, 1H, d  2 70.5 4.25  3 74.0 5.59  4 71.5 5.70  5 68.5 3.89  6 17.6 1.18, 3H, d Rh-3-Ang  1 167.2 —  2 127.9 —  3 138.7 5.92, 1 H, q  4 15.7 2.02, 3H, d  5 20.6 1.92, 3H, s Rh-4-Ang  1 167.2 —  2 128.0 —  3 137.9 5.87, 1 H, q  4 15.5 1.96, 3H, d  5 19.8 1.85, 3H, s 22-O-Ac  1 171.4 —  2 21.8 2.31, 3H, s

Based on these data and analysis, the structure of compound Y1 is assigned and shown below.

The chemical name of Y1 is: 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,16α, 21β, 22α, 28-pentahydroxyolean-12-ene.

Conclusion.

Based on the chemical shift analysis, the active compound Y1 isolated from extract of Xanthoceras Sorbifolia is a triterpenoid saponins with four sugars and biangeloyl groups attached to the sugar moiety. The formula of Y1 is C65H100027,

Results of Y2 Analysis

The profile of the proton NMR of Y2 is presented in FIG. 51. The profiles of 2D NMR (HMQC) of Y2 is presented in FIG. 52. Results of Y4 analysis The profile of the proton NMR of Y4 is presented in FIG. 53. The profiles of 2D NMR (HMQC) of Y4 is presented in FIG. 54.

Experiment 7 Acid and Alkaline and Enzyme Hydrolysis of Compound Y

Removal of Sugars from Compound Y.

Acid Hydrolysis of compound Y generates a compound with the following structure, designated herein as Y-c: (FIG. 37).

Methods:

5 mg of compound Y is dissolved in 3 ml of MeOH and then treated with 3 ml of 3N HCl. Hydrolysis of saponins will be conducted under reflux for 4 hr. After hydrolysis, the solution will be neutralized with 5% Na₂CO₃ and extracted with Ethyl acetate three times to afford an aqueous layer and an organic layer, containing sugars and aglycon, respectively. Aglycon from the organic layer will be further purified on Silica gel chromatography in (CHCl₃: MeOH, 1:9) or with C18 ODS HPLC chromatography. About 2 mg of compound with the above structure Y-c can be obtained.

Method reference: Essentials of Carbohydrate Chemistry. By John F. Robyt,

(Springer, 1998).

Partial Removal of Sugars from Compound Y.

The linkage of oligosaccharide can be cleaved by partial acid hydrolysis and by specific enzyme hydrolysis. For example, the 1→4 linkage of arabinofuranosyl can be removed by α-amylase. Other enzymes such as β-amylase, isoamylase, glucose oxidase, mannanse and pullulanase can be used to cleave individual saccharide in saponins.

Coumpound Structure showed in FIG. 70. Compound structure showed in FIG. 69. Compound Structure showed in FIG. 68.

Experiment 8 Purification of Component R from Xanthoceras Sorbifolia Extract

(A) Fractionation of Xanthoceras Sorbifolia extracts components with FPLC. Methods

Column: Octadecyl functionalized silica gel; column dimension: 2 cm×28 cm; equilibrated with 10% acetonitrile-0.005% TFA.

Sample loading: volume: 1-2 ml, concentration: 100 mg/ml in 10% acetonitrile/TFA. Gradient elution: 10-80% acetonitrile in a total volume of 500 ml.

Monitored at 254 nm.

Fraction Collector: 5 ml/fraction. Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.

Results

The elution profile shows 4-5 broad fractions (FIG. 20). These fractions were analyzed with HPLC. By comparison with the profiles of the original sample, specific component, in this case the R component, is identified and then collected for further purification.

Fractionation of R with FPLC with 30% Acetonitrile Isocratic Elution Methods

Column: Octadecyl-functionalized silica gel; column dimemsion: 2 cm×28 cm; equilibrated with 30% acetonitrile-0.005% TFA.

Sample loading: 0.2 ml, with concentration: 1-2 mg/ml. Elution: 30% acetonitrile isocratic. Monitor absorption wavelength: at 254 nm. Fraction Collector: 5 ml/fraction. Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.

Results

Fraction No. 39-41 from gradient elution of FPLC were pooled and further purified with an open ODS-C18 column with isocratic 30% acetonitrile elution. Six identifiable fractions in two groups were collected. See FIG. 45. Fractions 6-13 were further characterized with HPLC.

Analysis and Isolation of R with HPLC

Methods

Column: Waters μ-bondapak C18 (3.9×300 nm) and Waters DeltaPak C18 (7.8 mm×30 cm). Elution: Gradient (10-80%) and 30% isocratic elution. Flow rate: 0.5 ml/min; monitored at 207 nm; with attenuation 0.128; chart speed: 0.25 cm/min.

Results

On HPLC gradient elution analysis, Fractions #9-11 contain a major component with a few minor components. See FIG. 46. These components were further separated into 4-5 components with the 30% acetonitrile isocratic elution in a DeltaPak column. The fraction designated herein as “R1”, is the major component. See FIG. 47A. The pure R1 was subsequently collected from the column elution. See FIG. 47B.

Appearance and Solubility.

The pure R1 is amorphous white powder, soluble in aqueous alcohol (methanol, ethanol), 50% acetonitrile and 100% pyridine.

Determination of the chemical structure of R1 isolated from Xanthoceras Sorbifolia Extract

Methods

The NMR and MS Analysis of R1 are same as those described in Experiment 5.

Results

The proton NMR profile of pure R1 is presented in FIG. 21. The 2D NMR (HMQC) spectra of R1 are presented in FIG. 22. The 2D NMR (HMBC) spectra of R1 are presented in FIG. 23. The 2D COSY spectrum is presented in FIG. 24. The Carbon 13 NMR spectrum is presented in FIG. 25.

Based on all the data presented above, Table 8.1 summarizes the results of the structural analysis and the assignment of the functional groups of compound R1.

TABLE 8.1 ¹³C and ¹H NMR Data for R1 (in Pyridine-d₅)^(a) Position C H Key HMBC correlations 1 38.6 1.01, 1.63 C-3, C-25 2 26.4 1.89, 2.33 C-3 3 89.1 3.26, 1H, dd, 12.0/4.2 Hz C-23, C-24, Glc′ C-1 4 39.2 — — 5 55.5 0.69, 1H, d, 11.4 Hz C-4, C-6, C-7, C-9, C-10, C-23, C-24, C-25 6 18.3 1.30, 1.39 C-5, C-8, C-10 7 32.5 1.41 C-6, C-26 8 40.1 — — 9 47.7 1.61 C-1, C-5, C-8, C-10, C-11, C-14, C-25, C-26 10 36.7 — — 11 23.7 1.90, 2.00 C-8, C-12, C-14 12 123.5 5.35, 1H, br s C-9, C-14, C-18 13 142.9 — — 14 41.9 — — 15 25.7 1.88, 1.90 — 16 18.0 1.95, 2.29 C-14, C-17, C-18 17 43.1 — — 18 41.6 2.60, dd, 12.0/2.4 Hz C-19 19 46.3 1.28, 2.11 C-18, C-20, C-29, C-30 20 36.1 — — 21 76.5 3.73, 1H, d, 9.6 Hz C-20, C-22, C-29, C-30 22 75.1 4.31, 1H, d, 9.6 Hz C-16, C-17, C-21 23 27.9 1.20, 3H, s C-3, C-4, C-5, C-24 24 16.6 0.95, 3H, s C-3, C-4, C-5, C-23 25 15.7 0.95, 3H, s C-1, C-5, C-9, C-10 26 16.7 1.07, 3H, s C-7, C-8, C-9, C-14 27 26.1 1.25, 3H, s C-8, C-13, C-14 28 75.8 4.10, 2H, br s C-16, C-17, C-18, C-22, Glc′″ C-1 29 30.2 1.22, 3H, s C-19, C-20, C-21, C-30 30 19.5 1.26, 3H, s C-19, C-20, C-21, C-29 3-Glc′ 1 106.5 4.84, 1H, d, 7.2 Hz C-3, Glc′ C-5 2 73.2 3.99 — 3 79.1 5.90, 1H, t, 9.6 Hz Ang C-1, Glc′ C-2, C-4 4 69.4 4.26 Glc′ C-6 5 76.4 3.95 Glc′ C-1, C-3 6 69.8 4.40, 4.83 Glc′ C-4, Glc″ C-1 Ang 1 167.9 — — 2 128.7 — — 3 136.7 5.80, 1H, ddd, 6.6/0.6 Hz Ang C-1, C-4, C-5 4 15.7 1.93, 3H, dd, 6.6/0.6 Hz Ang C-2, C-3 5 20.6 1.81, 3H, s Ang C-1, C-2, C-3 Glc″ 1 105.4 5.09, 1H, d, 7.8 Hz Glc′ C-6, Glc″ C-5 2 74.9 4.05 Glc″ C-1 3 78.2^(b) 4.22 Glc″ C-5 4 71.4^(c) 4.24 Glc″ C-6 5 78.3^(d) 3.90 — 6 62.4^(e) 4.40, 4.51 Glc″ C-4 28-Glc′″ 1 103.5 4.72, 1H, d, 7.2 Hz C-28 2 75.3 4.22 Glc′″ C-1, C-3, Rha C-1 3 79.8 4.25 — 4 71.6 4.20 — 5 76.6 4.13 Glc′″ C-3 6 70.0 4.67, 2H, d, 10 Hz Glc″″ C-1, Glc′″ C-4, C-5 Rha 1 100.7 6.52, 1H, br s Glc′″ C-2, Rha C-3, C-5 2 72.3^(f) 4.70, d, 3.0 Hz Rha C-4 3 72.3^(f) 4.63, dd, Rha C-2 4 74.1 4.35 Rha C-2, C-5 5 69.1 4.79 — 6 18.7 1.82, 3H, d, 6.6 Hz Rha C-4, C-5 Glc″″ 1 105.5 5.01, 1H, d, 7.8 Hz Glc′″ C-6, Glc″″ C-5 2 74.9 4.05 Glc″″ C-1 3 78.2^(b) 4.22 Glc″″ C-5 4 71.4^(c) 4.24 Glc″″ C-6 5 78.3^(d) 3.90 — 6 62.5^(e) 4.40, 4.51 Glc″″ C-4 ^(a)The data were assigned based on COSY, HMQC and HMBC correlations. ^(b,c,d,f)The data with the same labels in each column were overlapped. ^(e)The data with the same labels in each column may be interchanged.

Conclusion

Based on the chemical shift analysis, the compound R1 isolated from extract of Xanthoceras Sorbifolia is a triterpenoid saponins with five sugars and one angeloyl group attached to the sugar moiety. The chemical structure of R1 is:

The formula of compound R1 is C₆₅H₁₀₆O₂₉, and the chemical name of R1 is: 3-O-[angeloyl-(1→3)-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β, 21β, 22α, 28-tetrahydroxyolean-12-ene

Experiment 9

Purification of Component-O from Xanthoceras Sorbifolia Extract

(A) Fractionation of Xanthoceras Sorbifolia Extracts Components with FPLC

Methods:

Column: Octadecyl functionalized silica gel; column dimension: 2 cm×28 cm; equilibrated with 10% acetonitrile-0.005% TFA.

Sample loading: 1-2 ml, concentration: 100 mg/ml in 10% acetonitrile/TFA. Gradient elution: 10-80% acetonitrile in a total volume of 500 ml. Monitor absorption wavelength: at 254 nm. Fraction Collector: 5 ml/fraction. Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.

Results.

The elution profile shows 4-5 broad fractions (FIG. 20). These fractions were analyzed with HPLC. By comparison with the profiles of the original sample, specific component, in this case the component-O, is identified (#28-30) and were collected for further purification.

(B) Purification of Component-O with HPLC with 20% Acetonitrile Isocratic Elution.

Methods.

Column: A preparative HPLC column (Waters Delta Pak C18-300A); Samples: Fraction #28-30 from the gradient elution of FPLC were pooled and applied into the HPLC. Elution: 20% acetonitrile isocratic with flow rate of 1 ml/min. Fractions were collected. Monitored at 207 nm; Fractions of interested were collected and lyophilized.

Results.

Sixteen identifiable fractions were observed in the elution profiles (FIG. 48). Fractions 28, 34 and 54 were further characterized with HPLC using same condition (FIGS. 49 and 50). As show in these figures, a single peak elution of fractions 28, 34 and 54 was obtained, indicating that they are homogeneous fractions (pure). These purified components are named as Compound O28, O34 and O54, respectively.

Appearance and solubility: The purified O-23 and O-34 are light yellow amorphous powder, soluble in aqueous alcohol (methanol, ethanol), 50% acetonitrile and 100% pyridine. The purified O-54 is white amorphous powder, soluble in aqueous alcohol (methanol, ethanol), 50% acetonitrile and 100% pyridine.

(C) Structure Analysis of Compound-O54. Methods:

The NMR and MS analysis of O54 are same as those described in Experiment 5.

Results

The profile of the proton NMR of compound-O54 is presented in FIG. 28. The 2D NMR (HMQC) spectra of O54 is presented in FIG. 29. The 2D NMR (HMBC) spectra of O54 are presented in FIG. 30.

Based on all the data presented above, Table 9.1 summarizes the results of the structural analysis and the assignment of the functional groups of compound-O54.

TABLE 9.1 ¹³C and ¹H NMR data for 054 (in Pyridine-d₅)^(a) Position C H Key HMBC correlations 1 38.6 1.03, 1.60 C-3, C-25 2 26.3 1.90, 2.40 C-3 3 89.1 3.30, 1H, dd, 12.0/4.8 Hz C-23, C-24, Glc′ C-1 4 39.3 — — 5 55.6 0.70, 1H, d, 12.0 Hz C-4, C-6, C-7, C-9, C-10, C-23, C-24, C-25 6 18.5 1.30, 1.40 C-5, C-8, C-10 7 32.5 1.40 C-6, C-26 8 40.2 — — 9 47.8 1.59 C-1, C-5, C-8, C-10, C-11, C-14, C-25, C-26 10 36.6 — — 11 23.6 1.90, 1.95 C-8, C-12, C-14 12 123.5 5.34, 1H, br s C-9, C-14, C-18 13 142.8 — — 14 41.9 — — 15 25.8 1.90 — 16 18.1 1.90, 2.31 C-14, C-17, C-18 17 43.2 — — 18 41.7 2.58, dd, 13.2/2.0 Hz C-19 19 46.5 1.28, 2.08 (t, 1H, t, 13.2 Hz) C-18, C-20, C-29, C-30 20 36.2 — — 21 76.6 3.73, 1H, d, 10.2 Hz C-20, C-22, C-29, C-30 22 75.1 4.31, 1H, d, 10.2 Hz C-16, C-17, C-21 23 27.9 1.22, 3H, s C-3, C-4, C-5, C-24 24 16.8 0.98, 3H, s C-3, C-4, C-5, C-23 25 15.8 0.95, 3H, s C-1, C-5, C-9, C-10 26 16.9 1.07, 3H, s C-7, C-8, C-9, C-14 27 26.1 1.26, 3H, s C-8, C-13, C-14 28 75.9 4.10, 2H, br s C-16, C-17, C-18, C-22, Glc′″ C-1 29 30.1 1.24, 3H, s C-19, C-20, C-21, C-30 30 19.6 1.26, 3H, s C-19, C-20, C-21, C-29 3-Glc′ 1 106.5 4.86, 1H, d, 7.8 Hz C-3, Glc′ C-5 2 74.9 3.99 — 3 78.2^(b) 4.20 — 4 70.8 4.36 — 5 76.5 3.95 — 6 70.5 4.40, 4.88 (d, 1H, 9.6 Hz) Glc″ C-1 Glc″ 1 105.5 5.12, 1H, d, 7.8 Hz Glc′ C-6, Glc″ C-5 2 75.0^(c) 4.03 Glc″ C-1 3 78.2^(b) 4.20 — 4 71.5^(d) 4.20 — 5 78.3^(e) 3.91 — 6 62.4^(f) 4.40, 4.48 Glc″ C-4, C-5 28-Glc′″ 1 103.6 4.72, 1H, d, 7.2 Hz C-28 2 75.3 4.22 Glc′″ C-1, C-3, Rha C-1 3 79.8 4.21 — 4 71.6 4.19 — 5 76.8 4.14 Glc′″ C-3 6 70.3 4.67, 2H, d, 10.2 Hz Glc″″ C-1, Glc′″ C-4, Rha C-5 1 100.6 6.51, 1H, br s Glc′″ C-2, Rha C-3, C-5 2 72.3^(g) 4.72, d, 3.0 Hz Rha C-4 3 72.3^(g) 4.61, dd, 10.8/3.6 Hz, Rha C-2 4 74.1 4.36 Rha C-2, C-5 5 69.2 4.77 — 6 18.6 1.80, 3H, d, 6.0 Hz Rha C-4, C-5 Glc″″ 1 105.6 5.00, 1H, d, 7.8 Hz Glc′″ C-6, Glc″″ C-5 2 75.0^(c) 4.03 Glc″″ C-1 3 78.2^(b) 4.20 — 4 71.5^(d) 4.20 — 5 78.3^(e) 3.91 — 6 62.5^(f) 4.40, 4.48 Glc″″ C-4, C-5 ^(a)The data were assigned based on COSY, HMQC and HMBC correlations. ^(b,c,d,e,g)The data with the same labels in each column were overlapped. ^(f)The data with the same labels in each column may be interchanged.

Conclusion

Based on the chemical shift analysis, the compound O54 isolated from extract of Xanthoceras Sorbifolia is a triterpenoid saponins with five sugars. The chemical structure of Compound O54 is:

The structure of compound O54 is presented in the following figure.

The formula of compound O54 is C₆₀H₁₀₀O₂₈, and the chemical name of O54 is: The chemical name of compound-O54 is: 3-O-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β, 21β, 22α, 28-tetrahydroxyolean-12-ene.

Results of O28 Analysis

The profile of the proton NMR of O28 is presented in FIG. 55.

The profiles of 2D NMR (HMQC) of O28 is presented in FIG. 56.

Results of O34 Analysis

The profile of the proton NMR of O34 is presented in FIG. 57.

The profiles of 2D NMR (HMQC) of O34 is presented in FIG. 58.

Experiment 11: Extracts X and Y from the Wenguanguo Plant (Xanthoceras Sorbifolia) Improve Learning in Normal Aging Mice

Extracts X and Y are Different Wenguanguo Extracts

Aging male mice at 16 months of age weighing 35-55 gm were trained in a SMG-2 filled with water 11 cm deep (25-26° C.). The SMG-2 had a start point, 4 blind terminals, the escape platform and their routes. The mice were trained to find the escape platform, and the escape latencies from the water and error frequencies were recorded. After training 3 times, the mice which escaped from the water in 2 minutes were selected for the test. The selected aging mice were divided into 9 groups of 11: 1) control: receiving normal saline (NS); 2) Positive control: receiving Xi-en-kai 0.9 g/kg; 3) X-I group: receiving 100 mg/kg4; 4) X-11 group: receiving 200 mg/kg; 5) X-III group: receiving 400 mg/kg; 6) Y-1 group: receiving 125 mg/kg; 7) Y-11 group: receiving 250 mg/kg; 8) Y-III group: receiving 500 mg/kg and 9) model group.

All the drugs were received through oral administration, 20 ml/kg, 3, 6 and 9 days before the water maze test. The escape latencies (EL) from the water and error frequencies were recorded. All data were analyzed with t-test.

11.1. After 3 days of administration of extracts X and Y from Wenguanguo Plant, hereinafter as “X” and “Y”, respectively, the escape latency in the water maze by the group 9 mice receiving 500 mg/kg of Y decreased significantly compared with the control (P<0.05). The other dosage treatments showed improvement too but not significant ones. See Table 11.1.

TABLE 11.1 The Learning Effects of Plant Extracts after Administration in Aging Mice for 3 days Blind Blind Blind Blind Error Group Terminal 1 Terminal 2 Terminal 3 Terminal 4 Frequency EL(sec) control 2.12 ± 2.27 3.62 ± 1.63 1.37 ± 2.41 2.68 ± 2.52  9.81 ± 3.42 64.12 ± 24.8 positive 1.09 ± 1.57  2.0 ± 0.89  2.0 ± 1.26  2.0 ± 2.64  7.09 ± 2.30 39.45 ± 16.32 X I 1.18 ± 1.4 3.36 ± 2.65 2.27 ± 2.14 0.81 ± 1.07  7.63 ± 4.47 51.72 ± 17.23 X II  2.2 ± 1.61  2.5 ± 1.51  2.0 ± 1.69  2.8 ± 1.03  9.5 ± 4.03  50.3 ± 20.84 X III 1.45 ± 2.33 2.72 ± 2.64 2.09 ± 2.11 1.81 ± 1.94  8.09 ± 4.67 46.91 ± 19.18 Y I 1.36 ± 1.91  3.0 ± 1.94 3.45 ± 3.2 1.09 ± 1.44  8.18 ± 3.78 46.36 ± 22.33 Y II 1.63 ± 1.80 3.81 ± 1.94 2.36 ± 1.12  .36 ± 1.50 10.18 ± 3.02 48.36 ± 20.61 Y III 2.18 ± 3.34 1.63 ± 1.21 1.54 ± 1.29 1.81 ± 1.40  7.18 ± 4.30 41.45 ± 16.48* P < 0.05*

11.2. After 6 days of administration of X and Y, the error frequency in the water maze in the mice with all dosage treatments decreased significantly (P<0.05, P<0.01). The escape latency in the water maze by the group 9 mice receiving 500 mg/kg of Y decreased significantly (P<0.05). See Table 11.2.

TABLE 11.2 The Learning Effects of Plant Extract after Administration in aging mice for 6 days Blind Blind Blind Blind Group Terminal Terminal Terminal Terminal Error Frequency EL(sec) Control 2.74 ± 2.98  4.0 ± 2.67 3.26 ± 3.02  3.0 ± 2.67 12.9 ± 4.20 60.58 ± 24.6 positive 1.64 ± 2.06 3.73 ± 2.05 1.18 ± 1.47  2.0 ± 1.90 8.55 ± 4.61* 38.64 ± 13.68 X I  1.7 ± 1.49  3.1 ± 2.02  1.8 ± 2.10  1.9 ± 1.66  8.5 ± 1.65*  47.7 ± 11.13 X II 1.18 ± 1.66  3.5 ± 2.02  2.0 ± 1.73 1.73 ± 2.05 8.45 ± 3.14*  49.0 ± 13.29 X III 1.09 ± 1.30 3.55 ± 2.07 1.91 ± 1.58 1.55 ± 1.44  8.0 ± 2.32* 46.36 ± 13.31 Y I  1.0 ± 1.67 3.09 ± 1.58 2.64 ± 1.8 1.54 ± 2.02 8.36 ± 3.07* 57.27 ± 19.88 Y II 1.36 ± 1.50 3.36 ± 2.06  2.0 ± 1.67 1.18 ± 1.17 7.91 ± 3.05** 47.55 ± 22.93 Y III  1.2 ± 1.14  4.1 ± 1.79  2.5 ± 2.84  1.4 ± 1.90  9.0 ± 3.23*  39.9 ± 8.56* P < 0.05* P < 0.01**

11.3. After 9 days of administration of X and Y, the error frequency in the water maze in the mice with all dosage treatments decreased significantly (P<0.05, P<0.01). The escape latency in the water maze by the group 9 mice receiving 500 mg/kg of Y decreased significantly (P<0.05). See Table 11.3. FIG. 60a, 60b .

TABLE 11.3 The Learning Effects of Plant Extracts after Administration in aging mice for 9 days Blind Blind Blind Blind Error Group Terminal 1 Terminal 2 Terminal 3 Terminal 4 Frequency EL(sec) Control 2.36 ± 1.65 2.64 ± 1.69 2.86 ± 2.54  1.5 ± 2.18 9.71 ± 3.52 59.71 ± 29.42 positive 1.64 ± 1.80 2.18 ± 1.54 1.55 ± 1.37 1.64 ± 2.20  7.0 ± 2.19* 33.36 ± 10.87 X I  1.1 ± 1.29  2.5 ± 2.37  1.9 ± 0.74  1.0 ± 1.15  6.5 ± 3.27*  40.8 ± 20.4 X II 1.18 ± 1.17 2.18 ± 1.60 1.36 ± 1.36 2.18 ± 1.25 6.91 ± 3.27* 42.73 ± 15.82 X III  1.0 ± 1.25  1.9 ± 1.79  1.3 ± 1.25  2.2 ± 1.16  6.4 ± 2.84*  35.1 ± 11.76* Y I 1.82 ± 1.33 1.64 ± 1.69 1.82 ± 1.33 1.82 ± 1.47 7.09 ± 2.47* 42.09 ± 20.93 Y II  1.2 ± 1.32  1.9 ± 1.37  2.6 ± 1.58  1.2 ± 1.32  7.1 ± 1.52*  34.4 ± 13.47* Y III  0.8 ± 1.03  2.5 ± 1.43  1.8 ± 1.40  2.0 ± 1.70  7.0 ± 1.41*  31.9 ± 9.87** P < 0.05* P < 0.01**

The results indicated that the extracts X and Y had positive effects on improving acquisition and retention of the tested aging mice. In addition, the effects increased with the period of receiving the extracts of X and Y prolonged Experiment 12: Effects of Extracts X and Y on Improving Impairment Induced by Pentobarbital Sodium in Water Maze Learning

12.1 After 10 days of administration of the extracts X and Y, the administrated mice were injected with pentobarbital sodium to induce amnesia.

After 1 day administration of pentobarbital sodium, the results of water maze learning showed that the time spent searching the terminal platform in the water maze by the mice receiving 100 mg/kg of X, and 125 mg, 250 mg/kg and 500 mg/kg of Y decreased significantly (P<0.05).

Error frequency made in the water maze by the mice receiving 500 mg/kg of Y decreased significantly (P<0.05). See Table 12.1.

TABLE 12.1 Results of Water Maze Learning (First Day after Injection with Pentobarbital) Blind Blind Blind Blind Error Group Terminal 1 Terminal 2 Terminal 3 Terminal 4 Frequency EL(sec) control 0.92 ± 0.76 1.46 ± 0.97 1.85 ± 1.07 1.23 ± 0.73 5.38 ± 2.33  63.0 ± 25.31 positive 0.69 ± 0.70  1.3 ± 0.95  1.1 ± 0.74  1.1 ± 0.74  4.1 ± 1.85  36.5 ± 15.76** X I  0.5 ± 0.53  1.7 ± 0.82  1.2 ± 0.92  0.9 ± 0.32  4.2 ± 1.62  42.2 ± 18.83* X II  0.9 ± 0.88  1.4 ± 0.70  1.6 ± 1.35  1.1 ± 0.88  5.0 ± 2.49  53.8 ± 16.10 X III  0.9 ± 0.74  1.7 ± 0.82  1.8 ± 0.42  0.9 ± 0.57  5.4 ± 1.58  58.1 ± 16.11 Y I  1.0 ± 0.89 1.36 ± 0.81 1.27 ± 1.01 0.73 ± 0.65 4.09 ± 2.02 42.73 ± 16.17* Y II  0.9 ± 0.74  1.7 ± 0.82*  1.0 ± 0.82*  0.6 ± 0.70*  4.2 ± 1.87  38.4 ± 15.19** Y III  0.6 ± 0.70  0.8 ± 0.63  1.4 ± 1.35  0.8 ± 0.63  3.6 ± 1.26*  38.5 ± 13.81** P < 0.05* P < 0.01**

12.2. After two days of injected of pentobarbital sodium, the time spent searching the terminal platform and the error frequency made in the water maze by all groups of mice receiving X and Y decreased significantly (P<0.05). See Table 12.2.

TABLE 12.2 Results of Water Maze Learning (Second Day after Injection with Pentobarbital) Blind Blind Blind Blind Error Group Terminal 1 Terminal 2 Terminal 3 Terminal 4 Frequency EL(sec) control 1.36 ± 0.50 1.86 ± 0.53 1.29 ± 1.07 1.14 ± 0.66 5.64 ± 1.91  59.5 ± 34.95 positive  0.8 ± 0.63  1.8 ± 0.42  1.1 ± 0.74  0.6 ± 0.70  4.3 ± 0.95*  34.7 ± 11.45* X I  0.9 ± 0.57*  1.0 ± 0.67**  1.2 ± 0.79  0.8 ± 0.63  3.9 ± 1.73*  34.5 ± 12.67* X II  0.8 ± 0.79*  1.5 ± 0.71  1.1 ± 0.88  0.5 ± 0.53*  3.8 ± 1.40*  35.9 ± 12.70* X III  1.0 ± 1.05  1.3 ± 0.48*  1.3 ± 0.82  0.5 ± 0.53*  4.1 ± 1.79*  36.1 ± 11.10* Y I 1.09 ± 0.94 1.45 ± 0.52 0.91 ± 0.83 0.73 ± 0.65 4.18 ± 1.08* 36.64 ± 14.38* Y II  0.9 ± 0.57*  1.3 ± 0.82  1.1 ± 0.88  0.8 ± 0.63  4.1 ± 1.45*  35.5 ± 14.27* Y III  0.8 ± 0.63*  0.9 ± 0.74**  0.9 ± 0.57  0.9 ± 0.57  3.4 ± 1.43**  32.1 ± 13.12* P < 0.05* P < 0.01**

12.3. After three days of administration of pentobarbital sodium, the time spent searching the terminal platform in the water maze by all groups of mice receiving X and Y decreased significantly (P<0.05). The error frequency made in the water maze by the mice receiving 250 mg/kg and 500 mg/kg of Y decreased significantly (P<0.05). See Table 12.3, FIG. 61a and FIG. 61b

TABLE 12.3 Results of Water Maze Learning (Third day after Injection with Pentobarbital) Blind Blind Blind Blind Error Group Terminal 1 Terminal 2 Terminal 3 Terminal 4 Frequency EL(sec) control 0.85 ± 0.99 1.0 ± 0.58 1.46 ± 1.05 0.62 ± 0.65 3.92 ± 1.75 48.92 ± 23.49 positive  0.4 ± 0.52 0.7 ± 0.48  0.9 ± 0.74  0.6 ± 0.70  2.6 ± 0.97*  28.4 ± 13.78 X I  0.6 ± 0.70 0.8 ± 0.63  0.6 ± 0.70*  0.8 ± 0.92  2.8 ± 1.69  28.0 ± 17.10* X II  0.4 ± 0.52 1.0 ± 0.47  1.0 ± 0.82  0.6 ± 0.84  3.0 ± 1.25  32.0 ± 12.36* X III  0.5 ± 0.71 0.8 ± 0.63  1.0 ± 0.82  0.9 ± 0.74  3.2 ± 1.23  31.8 ± 12.48* Y I  0.5 ± 0.53 0.7 ± 0.48  1.0 ± 0.67  0.6 ± 0.52  2.8 ± 0.63  31.0 ± 9.66* Y II  0.5 ± 0.53 0.6 ± 0.52  0.8 ± 0.42  0.6 ± 0.52  2.5 ± 0.53*  29.0 ± 7.80* Y III  0.3 ± 0.48 1.0 ± 0.47  0.9 ± 0.57  0.4 ± 0.52  2.6 ± 0.84*  30.2 ± 7.54* P < 0.05*

The results indicated that the extracts X and Y had distinct positive effects on improving spatial learning and retention impairment induced by pentobarbital sodium.

Experiment 13 Effects of Extracts X and Y on Improving Impairment Induced by Scopolamine Hydrobromide in Passive Avoidance

ICR mice weighing 16-20 gm were trained in a STT-2. A mouse was placed on the platform and the SDL were recorded. When the mouse stepped down and all four feet were on the grid, it received electric shock (36 V) immediately, and the EL was recorded. The mice with SDL and EL within 2-60 seconds were selected for the test. The selected aging mice were divided into 9 groups. Each group had 5 male and 5 female mice. All the drugs were received with oral administration, 20 ml/kg, 3, 6 and 9 days before the platform training. The SDL, El and error frequency (the times of receiving electric shocks) made in 5 minutes were recorded. After 10 days of administration all the groups of mice received Scopolamine hydrobromide by injection, 3 mg/kg. After 30 minutes of administration of Scopolamine hydrobromide the mice were trained on the platform and the training was repeated the next day. The performances of the mice in the training were recorded. The SDL, EL and error frequency were recorded. All data were analyzed with t-test.

13.1. After 9 days of administration of X and Y, the results of passive avoidance in aging mice showed that the EL and the error frequency made by the mice received the X and Y in all doses reduced. See Table 13.1.

TABLE 13.1 Passive Avoidance in Aging Mice after 9 days of Administration Group EL (sec) SDL (min) Error Frequency (in 5 min) control 6.8 ± 5.93 131.5 ± 106.50 2.5 ± 1.47 positive 5.3 ± 3.74 148.6 ± 81.26 1.7 ± 1.25 X I 7.0 ± 5.46 143.3 ± 19.77 1.8 ± 0.92 X II 6.2 ± 5.71 141.1 ± 109.89 2.0 ± 1.25 X III 4.9 ± 3.70 145.7 ± 107.0 1.9 ± 1.52 Y I 5.1 ± 3.95 153.0 ± 123.52 1.6 ± 1.33 Y II 3.9 ± 2.77 162.7 ± 108.92 1.8 ± 1.48 Y III 5.7 ± 3.12 159.4 ± 83.20 1.9 ± 0.93

13.2. After 10 days of administration of the X and Y, the tested mice were administrated with scopolamine. The results of passive avoidance in aging mice showed that the error frequency made in the passive avoidance by the mice receiving 400 mg/kg of X, 250 mg/kg and 500 mg/kg of Y reduced significantly (P<0.05, P<0.01). See Table 13.2.

TABLE 13.2 Effects of plant extract X and Y on improving impairment induced by Scopolamine Group EL (sec) Error Frequency (5 min) control 3.4 ± 3.03 0.9 ± 1.29 model 4.4 ± 4.09 1.6 ± 1.35 positive 3.1 ± 1.20 0.4 ± 0.52* X I 3.1 ± 2.08 0.8 ± 1.03 X II 3.7 ± 3.06 0.9 ± 1.52 X III 2.8 ± 1.48 0.3 ± 0.48** Y I 3.2 ± 2.49 0.7 ± 1.06 Y II 2.5 ± 0.97 0.5 ± 0.71* Y III 2.5 ± 0.71 0.5 ± 0.71* *p < 0.05 **p < 0.01

The results of passive avoidance test impaired mice by scopolamine showed that the error frequency made by the mice receiving X and Y in all doses reduced significantly (P<0.05). The SDL prolonged significantly in mice receiving 250 mg/kg of Y.

The results indicated that the extracts X and Y had distinct positive effects on improving learning and retention impairment induced by scopolamine. See Table 3.3.

TABLE 13.3 Effects of plant extract X and Y on improving impairment induced by Scopolamine. Group SDL (sec) Error Frequency (5 min) control 230.4 ± 96.61 0.7 ± 1.06 model 216.2 ± 100.77 1.5 ± 1.35 positive 286.0 ± 34.38* 0.4 ± 0.70* X I 245.7 ± 114.48 0.4 ± 0.84* X II 260.4 ± 87.14 0.4 ± 0.84* X III 266.8 ± 65.64 0.5 ± 0.71* Y I 252.7 ± 101.11 0.4 ± 0.84* Y II 285.8 ± 29.21* 0.4 ± 0.70* Y III 277.4 ± 47.62 0.4 ± 0.70* *p < 0.05

The results indicated that the extracts X and Y had distinct positive effects on improving learning and retention impairment induced by scopolamine hydrobromide.

Experiment 14 Effects of Extracts X and Y on Improving Impairment Induced by NaNO₂ in Water Maze Learning

ICR male mice weighing 16-19 gm were trained in a SMG-2 filled with water 11 cm deep (25-26° C.). The SMG-2 has a start point, 4 blind terminals, the escape platform and their routes. The mice were trained to find the escape platform, and the escape latencies from the water and error frequencies were recorded. After training, the mice which escaped from the water within 1 minute were selected for the test. The selected aging mice were divided into 9 groups of 11 mice. All the drugs were received with oral administration, 20 ml/kg, 3, 6 and 9 days before the water maze test. After 10 days of administration all the groups of mice were received with NaNO₂ by injection, 120 mg/kg. After 24 hours of administration of NaNO₂ the mice were trained to find the escape platform, and the escape latencies from the water and error frequencies made in 2 minutes were recorded. The administration of pentobarbital sodium continued for 3 days and the performances of the mice in water maze test were recorded. The escape latencies (EL) from the water and errors frequencies were recorded. All data were analyzed with t-test.

14.1. After 3 days of administration of X and Y the escape latency from the water maze and error frequency by the mice receiving X and Y reduced, although not significantly. See Table 14.1.

TABLE 14.1 Effects of Extracts X and Y on Water Maze Learning in Mice after 3 Days of Administration Blind Blind Blind Blind Group Terminal 1 Terminal 2 Terminal 3 Terminal 4 Error Frequency EL(sec) control 1.05 ± 0.49 1.82 ± 0.66 1.09 ± 0.75 0.86 ± 0.47 4.82 ± 0.96 48.27 ± 21.47 positive 0.91 ± 0.30 1.27 ± 0.65 0.82 ± 0.60 0.64 ± 0.50 3.64 ± 0.92** 36.27 ± 11.83 X I 1.36 ± 0.81 1.73 ± 0.79 1.09 ± 0.83 0.55 ± 0.52 4.73 ± 2.05 37.82 ± 14.24 X II 0.91 ± 0.30 1.82 ± 0.40 1.09 ± 0.94 1.00 ± 0.89 4.91 ± 1.51 36.46 ± 11.97 X III 1.09 ± 0.54 1.45 ± 0.52 0.91 ± 0.70 0.45 ± 0.52 3.91 ± 0.70 36.46 ± 11.78 Y I 1.55 ± 0.52 1.82 ± 0.40  1.0 ± 0.89 0.45 ± 0.69 4.82 ± 1.33 41.46 ± 16.37 Y II 0.91 ± 0.30 1.18 ± 0.60 1.27 ± 1.10 0.73 ± 0.79 4.09 ± 2.21 36.82 ± 20.61 Y III 0.91 ± 0.30 1.55 ± 0.82 0.45 ± 0.52 0.82 ± 0.40 3.73 ± 1.27 37.55 ± 13.85 *P < 0.05 **P < 0.01

14.2. After 6 days of administration of X and Y the escape latency from the water maze by the mice receiving 400 mg/kg of X and 500 mg/kg of Y reduced significantly compared with the control (P<0.01). See Table 14.2.

TABLE 14.2 Effects of Extracts X and Y on Water Maze Learning in Mice after 6 Daysof Administration Blind Blind Blind Blind Error Group Terminal 1 Terminal 2 Terminal 3 Terminal 4 Frequency EL(sec) control 0.95 ± 0.38 1.09 ± 0.43 0.77 ± 0.61 0.91 ± 0.53 3.82 ± 0.80 42.96 ± 13.48 positive 0.73 ± 0.65 1.09 ± 0.30 0.55 ± 0.52 0.82 ± 0.40 3.18 ± 0.75* 32.91 ± 7.15* X I 0.73 ± 0.65 1.27 ± 0.47 0.73 ± 0.65 0.73 ± 0.65 3.45 ± 1.21 37.18 ± 7.65 X II 0.91 ± 0.30 1.00 ± 0.45 0.64 ± 0.92 0.91 ± 0.54 3.45 ± 1.21 37.73 ± 13.26 X III 0.91 ± 0.30 1.09 ± 0.54 0.91 ± 0.83 0.82 ± 0.75 3.73 ± 1.19 31.09 ± 8.15** Y I 0.91 ± 0.30  1.0 ± 0.45 0.82 ± 0.40 0.55 ± 0.52 3.36 ± 0.67 35.82 ± 9.93 Y II 0.91 ± 0.54 1.09 ± 0.54 1.00 ± 0.77 0.64 ± 0.67 3.64 ± 1.12 35.09 ± 12.13 Y III 0.82 ± 0.40 1.09 ± 0.54 0.82 ± 0.60 0.55 ± 0.52 3.27 ± 1.10 31.73 ± 8.36** *P < 0.05 **P < 0.01

14.3. After 9 days of administration of X and Y, the escape latency from the water maze by the mice receiving 250 mg/kg of X, 250 mg and 500 mg/kg of Y reduced significantly compared with the control (P<0.05). See Table 14.3.

Tablet 14.3. Effects of Extracts X and Y on Water Maze Learning in Mice after 9 Days of Administration. Blind Blind Blind Blind Group Terminal 1 Terminal 2 Terminal 3 Terminal 4 Error Frequency EL(sec) control 0.59 ± 0.73 1.14 ± 0.64 0.55 ± 0.67  1.0 ± 0.76 3.27 ± 1.32 39.27 ± 15.52 positive 0.55 ± 0.52 1.00 ± 0.00 0.27 ± 0.65 0.91 ± 0.54 2.73 ± 0.65 27.64 ± 6.96* X I 0.45 ± 0.52 1.27 ± 0.47 0.73 ± 0.65 0.55 ± 0.52 3.00 ± 0.77 33.55 ± 9.59 X II 0.45 ± 0.52 0.91 ± 0.70 0.55 ± 0.69 0.82 ± 0.89 2.73 ± 0.90 28.00 ± 9.53* X III 0.45 ± 0.52 1.09 ± 0.70 0.82 ± 0.75 0.45 ± 0.52 2.82 ± 1.25 29.45 ± 8.49 Y I 0.91 ± 0.70 0.91 ± 0.54 0.45 ± 0.52 0.55 ± 0.52 2.82 ± 0.98 32.00 ± 9.49 Y II 0.64 ± 0.50 0.82 ± 0.75 0.64 ± 0.67 0.82 ± 0.60 2.91 ± 1.30 26.36 ± 9.82* Y III 0.73 ± 1.01 0.91 ± 0.30 0.45 ± 0.69 0.55 ± 0.69 2.64 ± 1.12 28.09 ± 9.26* *P < 0.05

14.4. After 10 days of administration of X and Y, the error frequency made in the water maze by the mice receiving 250 mg and 500 mg/kg of Y, the escape latency from the water maze by the mice receiving 500 mg/kg of Y reduced significantly compared with the control (P<0.05, P<0.01). See Table 14.4.

TABLE 14.4 Effects of Extracts X and Y on Water Maze Learning in Mice after 10 Days of Administration Blind Blind Blind Blind Error Group Terminal 1 Terminal 2 Terminal 3 Terminal 4 Frequency EL(sec) control 0.64 ± 0.73 1.14 ± 0.56 0.64 ± 0.66 0.91 ± 0.68 3.32 ± 0.95 36.73 ± 13.02 positive 0.45 ± 0.69 0.82 ± 0.40 0.55 ± 0.52 0.82 ± 0.40 2.64 ± 0.81*  29.0 ± 10.10 X I 0.36 ± 0.67 0.91 ± 0.70 0.91 ± 0.83 0.64 ± 0.81 2.82 ± 1.08 33.09 ± 13.96 X II 0.73 ± 0.79 0.82 ± 0.40 0.55 ± 0.69 0.73 ± 0.47 2.82 ± 0.75 28.91 ± 13.34 X III 0.91 ± 0.70 0.91 ± 0.54 0.73 ± 0.65 0.36 ± 0.67 2.91 ± 0.94 32.45 ± 13.57 Y I 0.73 ± 0.79 0.91 ± 0.30 0.36 ± 0.50 0.73 ± 0.65 2.73 ± 0.90 29.55 ± 13.87 Y II 0.55 ± 0.52 0.64 ± 0.50 0.45 ± 0.52 0.64 ± 0.67 2.27 ± 0.79** 30.36 ± 12.30 Y III 0.45 ± 0.69 1.09 ± 0.54 0.27 ± 0.65 0.55 ± 0.52 2.36 ± 1.21* 25.64 ± 11.02* *P < 0.05 **P < 0.01

The results indicated that the extracts X and Y had distinct positive effects on improving the learning and retention in mice in a water maze. In addition, the effects increased with the period of receiving the extracts of X and Y prolonged.

14.5. After 10 days of receiving X and Y, the mice were administrated with Na NO2 after the test. The results of treatment with X and Y to prevent impairments induced by Na NO2 in water maze learning in aging mice showed that error frequency made by the mice receiving 100 mg/ka and 200 mg/kg of X, and Y of all doses reduced significantly (P<0.05). See Table 14.5. It indicated the extracts X and Y had distinct positive effects on preventing the impairments induced by Na NO₂. See Table 14.5.

TABLE 14.5 Effects of Extract X and Y on preventing Impairments Induced by NaNO₂ Blind Blind Blind Blind Group Terminal 1 Terminal 2 Terminal 3 Terminal 4 Error Frequency EL(sec) Control 0.27 ± 0.47 0.91 ± 0.54 0.55 ± 0.52 1.09 ± 0.54 2.82 ± 0.75 30.91 ± 12.36 Model 1.18 ± 0.75 0.91 ± 0.30 0.45 ± 0.52 0.73 ± 0.65 3.27 ± 1.01 36.45 ± 16.89 Positive 0.45 ± 0.52 0.91 ± 0.30 0.55 ± 0.52 0.45 ± 0.52 2.36 ± 0.81* 32.00 ± 15.83 X I 0.55 ± 0.52 0.82 ± 0.60 0.18 ± 0.40 0.82 ± 0.60 2.36 ± 0.81* 29.09 ± 13.80 X II 0.18 ± 0.40 0.82 ± 0.60 0.55 ± 0.52 0.82 ± 0.75 2.36 ± 0.92* 25.82 ± 10.82 X III 0.45 ± 0.52 0.64 ± 0.50 0.82 ± 0.40 0.91 ± 0.70 2.82 ± 1.33 31.09 ± 11.76 Y I 0.27 ± 0.47 0.91 ± 0.30 0.36 ± 0.50 0.73 ± 0.47 2.27 ± 0.79* 27.00 ± 10.73 Y II 0.45 ± 0.52 0.64 ± 0.50 0.36 ± 0.50 0.91 ± 0.30 2.36 ± 0.81* 25.82 ± 11.43 Y III 0.64 ± 0.50 0.82 ± 0.40 0.36 ± 0.50 0.64 ± 0.50 2.45 ± 0.82* 25.09 ± 9.67 *P < 0.05 **P < 0.01

Experiment 15 Effects of Wenguanguo (Xanthoceras sorbifolia) Extracts on Urination in Mice

As used herein, Extracts (or Fractions) X and Y are different extracts of Wenguanguo plant or Xanthoceras sorbifolia.

Methods of Experiment. One hundred twelve male ICR mice weighing 18-22 gm were divided into 8 groups of 14: 1, control: receiving normal saline (NS); 2, DCT group: receiving DCT 33.4 mg/kg; 3, X-I group: receiving 100 mg/kg4; 4, X-II group: receiving 200 mg/kg; 5, X-III receiving 400 mg/kg; 6, Y-I group: receiving 125 mg/kg; 7, Y-II group: receiving 250 mg/kg and 8, Y-III group: receiving 500 mg/kg.

All the drugs were received with oral administration, 20 ml/kg, once a day for 3 days. After the last administration, the mouse was placed on a filter paper. The filter paper was on the bottom of a 500 ml beak. The quantity of urine was measured at 30, 60, 120, 180, 240, 300, and 360 minutes by weighing the filter paper with the electronic analytical scale. All data were analyzed with t-test.

Results.

After 3 days of administration of X and Y the quantity of urine discharged at 30 minute by the mice receiving 400 mg/kg of X decreased significantly (P<0.01) compared with the mice receiving normal saline. The quantity of urine discharged at 60 minute by the mice receiving 600 mg/kg of Y decreased significantly compared with the mice receiving normal saline (P<0.01).

The quantity of urine discharged at 180 minutes by the mice receiving 200 mg/kg of X, 125 mg and 500 mg/kg of Y decreased significantly compared with the mice receiving normal saline (P<0.01). See Table 15.1.

TABLE 15.1 Effects of Extracts X and Y on the Quantity of Urine in Mice after 3 Days of Administration Group 30 min 60 min 120 min Control 0.267 ± 0.105 0.367 ± 0.162 0.382 ± 0.109 Positive 0.348 ± 0.06* 0.471 ± 0.169** 0.574 ± 0.249 X I 0.304 ± 0.072 0.274 ± 0.076 0.323 ± 0.173 X II 0.341 ± 0.107 0.323 ± 0.102 0.404 ± 0.138 X III 0.155 ± 0.056** 0.200 ± 0.140** 0.455 ± 0.211 Y I 0.216 ± 0.130 0.309 ± 0.093 0.341 ± 0.061 Y II 0.278 ± 0.063 0.278 ± 0.119 0.437 ± 0.112 Y III 0.227 ± 0.058 0.235 ± 0.035** 0.425 ± 0.133 Compared with the control group: P < 0.05* P < 0.01**

TABLE 15.1 Effects of Extracts X and Y on the Quantity of Urine in Mice After 3 Days of Administration (continued) Group 180 min 240 min 300 min 360 min Control 0.191 ± 0.080 0.161 ± 0.083 0.116 ± 0.06 0.103 ± 0.057 Positive 0.272 ± 0.131 0.182 ± 0.096 0.110 ± 0.051 0.085 ± 0.031 X I 0.184 ± 0.105 0.154 ± 0.093 0.124 ± 0.091 0.102 ± 0.064 X II 0.336 ± 0.103** 0.163 ± 0.10 0.107 ± 0.076 0.106 ± 0.075 X III 0.207 ± 0.112 0.204 ± 0.088 0.150 ± 0.066 0.116 ± 0.077 Y I 0.367 ± 0.104** 0.171 ± 0.085 0.173 ± 0.068* 0.093 ± 0.053 Y II 0.275 ± 0.206 0.145 ± 0.029 0.109 ± 0.036 0.106 ± 0.045 Y III 0.319 ± 0.086** 0.264 ± 0.114** 0.152 ± 0.084 0.135 ± 0.051 Compared with the control P < 0.05* and P < 0.01**

The results indicated that the extract X and Y can regulate the quantity of urine after 3 days of administration of X and Y.

After 5 days of administration of X and Y, the quantity of urine discharged at 30 minute by the mice receiving 400 mg/kg of X and 500 mg/kg of Y decreased, but not significantly compared with the mice receiving normal saline. The quantity of urine discharged at 4 hour by the mice receiving 400 mg/kg of X and Y in all doses increased significantly compared with the mice receiving normal saline (P<0.05, P<0.01). See Table 15.2.

TABLE 15.2 Effects of Extracts X and Y on the Quantity of Urine in Mice after 5 Days of Administration Group 30 min 60 min 120 min Control 0.327 ± 0.148 0.330 ± 0.194 0.291 ± 0.146 Positive 0.524 ± 0.206** 0.478 ± 0.185* 0.472 ± 0.292* X I 0.382 ± 0.138 0.251 ± 0.071 0.265 ± 0.172 X II 0.348 ± 0.144 0.324 ± 0.113 0.277 ± 0.131 X III 0.245 ± 0.167 0.236 ± 0.129 0.251 ± 0.142 Y I 0.331 ± 0.098 0.340 ± 0.133 0.291 ± 0.081 Y II 0.357 ± 0.130 0.290 ± 0.145 0.327 ± 0.157 Y III 0.230 ± 0.121 0.307 ± 0.082 0.363 ± 0.100 Compared with the control P < 0.05* and P < 0.01**

TABLE 15.2 Effects of Extracts X and Y on the Quantity of Urine in Mice After 5 Days of Administration (continued) Group 180 min 240 min 300 min 360 min Control 0.186 ± 0.086 0.117 ± 0.069 0.105 ± 0.06 0.104 ± 0.08 Positive 0.214 ± 0.151 0.110 ± 0.045 0.126 ± 0.056 0.112 ± 0.065 X I 0.188 ± 0.097 0.175 ± 0.088 0.177 ± 0.102* 0.133 ± 0.092 X II 0.258 ± 0.143 0.150 ± 0.077 0.167 ± 0.097* 0.130 ± 0.094 X III 0.226 ± 0.107 0.233 ± 0.132** 0.120 ± 0.059 0.125 ± 0.048 Y I 0.273 ± 0.156 0.215 ± 0.095** 0.166 ± 0.151 0.116 ± 0.068 Y II 0.181 ± 0.088 0.181 ± 0.089* 0.151 ± 0.104 0.101 ± 0.042 Y III 0.193 ± 0.09 0.217 ± 0.092** 0.112 ± 0.056 0.117 ± 0.043 Compared with the control P < 0.05* and P < 0.01**

The results indicated that the extract X and Y can regulate the quantity of urine after 5 days of administration of X and Y.

After 7 days of administration of X and Y, the quantity of urine was measured at 30, 60, 120, 180, 240, 300, and 360 minutes. The quantity of urine discharged at 30 minute by the mice receiving 200, 400 mg/kg of X and 250, 500 mg/kg of Y decreased significantly (P<0.05) but increased at 240 minutes compared with the mice receiving normal saline. See Table 15.3.

TABLE 15.3 Effects of Extracts X and Y on the Quantity of Urine in Mice after 7 Days of Administration Group 30 min 60 min 120 min Control 0.252 ± 0.142 0.347 ± 0.159 0.430 ± 0.192 Positive 0.434 ± 0.230* 0.606 ± 0.214** 0.590 ± 0.333 X I 0.301 ± 0.152 0.314 ± 0.149 0.342 ± 0.186 X II 0.291 ± 0.161 0.332 ± 0.135 0.285 ± 0.173* X III 0.212 ± 0.113 0.260 ± 0.103 0.309 ± 0.117* Y I 0.254 ± 0.175 0.283 ± 0.137 0.313 ± 0.178 Y II 0.261 ± 0.189 0.292 ± 0.129 0.300 ± 0.128* Y III 0.246 ± 0.170 0.268 ± 0.240 0.281 ± 0.146* Compared with the control P < 0.05* and P < 0.01**

TABLE 15.3 Effects of Extracts X and Y on the Quantity of Urine in Mice after 7 Days of Administration (continued) Group 180 min 240 min 300 min 360 min Control 0.285 ± 0.136 0.155 ± 0.119 0.122 ± 0.071 0.111 ± 0.061 Positive 0.314 ± 0.119 0.279 ± 0.192* 0.163 ± 0.087 0.148 ± 0.071 X I 0.267 ± 0.179 0.200 ± 0.114 0.176 ± 0.147 0.157 ± 0.077 X II 0.250 ± 0.116 0.203 ± 0.134 0.180 ± 0.079* 0.129 ± 0.085 X III 0.293 ± 0.142 0.250 ± 0.116* 0.194 ± 0.104* 0.151 ± 0.076 Y I 0.310 ± 0.168 0.248 ± 0.178 0.155 ± 0.108 0.113 ± 0.05 Y II 0.334 ± 0.208 0.259 ± 0.205 0.205 ± 0.109* 0.188 ± 0.113* Y III 0.267 ± 0.133 0.212 ± 0.125 0.205 ± 0.119* 0.169 ± 0.073* Compared with the control P < 0.05* and P < 0.01**

The results indicated that the extract X and Y can regulate the quantity of urine after 7 days of administration of X and Y.

After 10 days of administration of X and Y, the quantity of urine discharged at 120 minutes by the mice receiving 200, 400 mg/kg of X and 250, 500 mg/kg of Y decreased significantly (P<0.05) compared with the mice receiving normal saline. See FIG. 59.

TABLE 15.4 Effects of Extracts X and Y on the Quantity of Urine in Mice after 10 Days of Administration Group 30 min 60 min 120 min Control 0.292 ± 0.184 0.323 ± 0.158 0.418 ± 0.221 Positive 0.374 ± 0.159 0.432 ± 0.163* 0.643 ± 0.181** X I 0.306 ± 0.124 0.317 ± 0.088 0.339 ± 0.145 X II 0.292 ± 0.082 0.343 ± 0.120 0.279 ± 0.118* X III 0.266 ± 0.116 0.348 ± 0.161 0.274 ± 0.111* Y I 0.273 ± 0.117 0.331 ± 0.103 0.406 ± 0.175 Y II 0.289 ± 0.126 0.344 ± 0.147 0.254 ± 0.102* Y III 0.227 ± 0.129 0.322 ± 0.162 0.255 ± 0.124* Compared with the control P < 0.05* and P < 0.01**

TABLE 15.4 Effects of Extracts X and Y on the Quantity of Urine in Mice after 10 Days of Administration (continued) Group 180 min 240 min 300 min 360 min Control 0.203 ± 0.087 0.144 ± 0.098 0.108 ± 0.074 0.091 ± 0.060 Positive 0.253 ± 0.116 0.147 ± 0.067 0.095 ± 0.094 0.068 ± 0.049 X I 0.249 ± 0.094 0.172 ± 0.093 0.120 ± 0.058 0.093 ± 0.050 X II 0.225 ± 0.074 0.163 ± 0.051 0.116 ± 0.052 0.093 ± 0.051 X III 0.247 ± 0.104 0.186 ± 0.102 0.121 ± 0.053 0.098 ± 0.065 Y I 0.243 ± 0.101 0.171 ± 0.098 0.126 ± 0.086 0.098 ± 0.058 Y II 0.229 ± 0.097 0.164 ± 0.091 0.124 ± 0.094 0.111 ± 0.067 Y III 0.213 ± 0.102 0.170 ± 0.081 0.121 ± 0.059 0.095 ± 0.045 Compared with the control P < 0.05* and P < 0.01**

The results indicated that the extract X and Y can regulate the quantity of urine after 10 days of administration of X and Y.

Conclusion.

The results indicated that the extract X and Y can regulate the quantity of urine after 3-10 days of administration of X and Y.

Experiment 15A Antidiuresis Test of Rat in Metabolism Cage

The antidiuresis tests using Xanthoceras Sorbifolia Extract FS(X) and Xanthoceras Sorbifolia saponin extract FS(Y) were carried out in rats. Note: Extracts FS(X) and FS(Y) similar to Extracts X and Y respectively. FS(Y) and FS(X) are crude saponin extracts and the “FS(Y)” and “FS(X)” are the extract names that were used in the experiments.

SD rats were treated with FS(X) by oral administration, daily at the doses of 100, 200, 400 mg/kg for 25 days. The experiment included a negative control group and positive control. Collecting urine in Standard metabolism cage and observation indicators, including urine volume, urine Na+, K+, Cl− content, pH and osmotic pressure. The results obtained were as follows: (1) Extract FS(X) has dose-dependant antidiuresis effect. There is significant antidiuresis at all the time phases for 200 and 400 mg/kg. However, by urination rate, the 400 mg/kg dosage group has a more significant effect in that it can delay urination output by two hours. In addition, there is no change in the impact on total urine volume output. (2) The extract FS(X) increased the concentration of ions in urine after drug treatment. However, there is no apparent dose dependency. (3) FS (X) and FS (Y) have no significant impact on the pH value and osmotic pressure, but have slight impact on the specific gravity.

Objective.

Standard metabolism cage method was carried out in rats to research the antidiuresis of FS(X) and FS(Y).

Test Drug.

FS(X) is compositions of Xanthoceras Sorbifolia extract. FS(Y) is compositions of Xanthoceras Sorbifolia extract. The test drug is a suspension formulated with 0.5% sodium carboxymethyl cellulose (0.5% CMC-Na).

Test Animals.

Male SD rats, initial weight 150-200 g, 100 individuals. The test animals are raised in cages (volume: 20×30×45 cm), and each cage has 5 animals. The basal feed is the full-rate pellet feed for experimental rat, self-made by the Experimental Animal Center. The cage bottom padding is wood shaving and chaff, dried before use. After the padding replacement that is made every other day on average, the cages are disinfected before reuse. Laboratory temperature 23±2° C., humidity 40˜70%, with air-conditioning, exhaust and ventilation equipment, natural lighting and a light-shade cycle of about 12 hours.

Animal Screening.

The animals are placed individually in the cage for adaptation once a day for 6-10 hours per day for 2 days. Before placing them into the case, press the lower abdomen gently to discharge the remaining urine, inject 38° C. distilled water into the stomach at the volume of 25 ml/kg as the water load. Collect the urine in the metabolism cage within 2 hours after stomach injection; collect the remaining urine by pressing the lower abdomen gently immediately before taking them out of the cage. Animals whose urine volume attains above 40% of the injection volume will be qualified ones.

Test Instruments.

Standard metabolism cage, Automatic urine analyzer (Miditron Junior II), Urine osmotic pressure tester, and Urine ion tester (EL-ISE, Beckman).

Methods

Test groups.

There are 3 groups for the FS (X), i.e., for 100, 200, 400 mg/kg/day, respectively. There is a dosage group for FS (Y): 400 mg/kg/day; once a day, fed at 30 min after administration, for 25 days. Control group were fed with 0.5% CMC-Na into the stomach every day. The positive control drug is pituitrin, injected into the abdominal cavity at the rate of 0.25 u/kg before the animals are placed in the cage. Each group includes 10 animals.

Urine Collection and Observation Indicators.

For the test groups, the animals that have experienced the administration period are on diet for 18 hours before urine collection without prohibiting drinking. After pressing their lower abdomen gently to discharge the remaining urine, apply the water load of 38° C. distilled water at the volume of 50 ml/kg, and place them in the cage for urine collection. Collect the urine volume and times at 0.25 hr, 0.5 hr, 1 hr, 2 hr, 3 hr, 4 hr, 5 hr and 6 hr after water feeding, when the animals are taken out of the cage at 6 hr, press their lower abdomen gently to collect the remaining urine in the bladder. Conduct the routine urine examination (pH, erythrocyte, leucocyte, protein, etc); measure urine Na+, K+, Cl− concentrations and urine osmotic pressure for the urine samples.

Data Processing.

Urination speed, Relative urine volume, Urine Na+, K+, Cl− content data are shown in X±Sd, and are compared with the control group and subject to a student t-test.

Results Impact on Urine Volume.

FS (X) has dose-dependant antidiuresis effects. There is a significant antidiuresis effect at all the time phases using dosages of 200 and 400 mg/kg. See Table 15A-1, FIGS. 63 and 63A. However, by urination discharge rate, the 400 mg/kg group has a more significant effect in reducing the urine output during the first 2 hrs as compared with the control. See Table 15A-2, FIGS. 64 and 64A. The FS (Y) 400 mg/kg group has an equivalent drug efficacy to FX (X) 400 mg/kg during the first 2 hours of experiment. In addition, there is no apparent dose dependency in the impact on total urine volume during the whole experiment. The 400 mg/kg groups of FS(X) can significantly reduce the urine volume during the first 6 hours after the drug-treatment which is more effective than the positive control drug (pituitrin).

Impact on Na+, K+, Cl− Ions in Urine.

By reducing the urine volume, the test drug has increased the concentration of ions in urine to different extent. However, there is no apparent dose dependency. By ionic, there is almost no impact on Na+ and Cl− ions, there is a certain discharge promotion action on K+ ions. The concentration and discharge amount of all ions in urine from the positive drug have risen apparently.

Impact on Urine pH and Osmotic Pressure.

FS (X) and FS (Y) have no significant impact on the pH value and osmotic pressure, but have slight impact on the specific gravity.

FIGS. 63 and 63A, Table 15A-1, shows results of the urine volume with water load after FS(X) Xanthoceras Sorbifolia Extract administration for 25 days. FIGS. 64 and 64A, Table 15A-2, shows results of discharging urine speed with water load after FS(X) Xanthoceras Sorbifolia Extract administration for 25 days. FIG. 65, Table 15A-3, shows results of urine specific gravity and pH with water load after FS(X) Xanthoceras Sorbifolia Extract administration for 25 days. FIG. 66, Table 15A-4, Concentration of Na+, K+ and Cl− in urine with water load after FS(X) Xanthoceras Sorbifolia Extract administration for 25 days.

Although the present invention has been described in detail with particular reference to preferred embodiments thereof, it should be understood that the invention is capable of other different embodiments, and its details are capable of modifications in various obvious aspects. As is readily apparent to those skilled in the art, variations and modifications can be affected while remaining within the spirit and scope of the invention. Accordingly, the foregoing disclosure, description, and figures are for illustrative purpose only, and do not in any way limit the invention which is defined only by the claims.

TABLE 5.2 Chemical Shift Data of 2D NMR chemical shift of HMQC analysis of compound Y DU = C:/Bruker/XWIN-NMR, USER = guest, NAME = chan, EXPNO = 5, PROCNO = 1 F1PLO = 144.360 ppm, F1PHI = 10.797 ppm, F2PLO = 7.966 ppm, F2PHI = 0.417 ppm MI = 1.00 cm, MAXI = 10000.00 cm, PC = 1.400 ADDRESS FREQUENCY row [Hz]F1 [PPM]F1 # col [Hz]F2 [PPM]F2 INTENSITY 1 148 20698.986 137.1608 384 3574.377 5.9557 2.00 2 152 20564.195 136.2676 401 3471.337 5.7840 2.67 3 157 20465.209 135.6117 220 4533.779 7.5543 45.24 4 223 18893.424 125.1963 431 3295.261 5.4906 6.22 5 234 18649.311 123.5787 258 4311.820 7.1845 100.00 6 315 16736.119 110.9011 376 3620.289 6.0322 7.49 7 353 15834.069 104.9237 493 2934.550 4.8896 3.98 8 355 15778.398 104.5548 449 3192.387 5.3192 2.99 9 355 15778.398 104.5548 492 2936.414 4.8927 1.18 10 451 13524.788 89.6213 660 1951.827 3.2522 2.41 11 451 13524.788 89.6213 663 1934.401 3.2231 2.60 12 473 12994.274 86.1059 563 2520.987 4.2005 1.93 13 479 12861.933 85.2290 500 2891.933 4.8186 3.96 14 491 12583.008 83.3807 487 2967.012 4.9437 6.95 15 523 11826.204 78.3658 307 4025.464 6.7073 3.92 16 523 11826.204 78.3658 309 4011.801 6.6846 4.78 17 523 11826.204 78.3658 545 2627.492 4.3780 1.99 18 529 11690.020 77.4633 504 2866.164 4.7757 4.60 19 532 11624.016 77.0260 530 2713.960 4.5221 2.32 20 532 11624.016 77.0260 532 2703.563 4.5047 2.51 21 535 11536.339 76.4450 590 2363.678 3.9384 3.34 22 545 11299.475 74.8754 573 2461.387 4.1012 2.35 23 545 11299.475 74.8754 576 2447.179 4.0775 2.10 24 555 11063.554 73.3121 541 2653.693 4.4216 6.62 25 567 10795.113 71.5333 537 2673.042 4.4539 1.84 26 567 10795.113 71.5333 539 2662.683 4.4366 2.82 27 567 10795.113 71.5333 541 2650.933 4.4170 1.72 28 579 10495.725 69.5494 527 2734.037 4.5555 5.06 29 594 10156.363 67.3006 563 2523.651 4.2050 7.18 30 622 9486.037 62.8588 608 2256.690 3.7601 2.91 31 622 9486.037 62.8588 611 2241.336 3.7346 2.67 32 622 9486.037 62.8588 635 2100.199 3.4994 2.96 33 622 9486.037 62.8588 637 2086.756 3.4770 3.42 34 627 9381.439 62.1656 552 2586.967 4.3105 3.58 35 627 9381.439 62.1656 555 2568.334 4.2794 3.42 36 627 9381.439 62.1656 568 2494.546 4.1565 2.54 37 627 9381.439 62.1656 571 2474.559 4.1232 2.51 38 630 9297.809 61.6115 531 2709.734 4.5150 2.61 39 630 9297.809 61.6115 539 2660.734 4.4334 2.66 40 670 8349.676 55.3287 911 480.283 0.8003 2.14 41 670 8349.676 55.3287 913 465.738 0.7760 2.18 42 726 7038.860 46.6427 679 1839.659 3.0653 2.51 43 726 7038.860 46.6427 819 1016.627 1.6939 1.42 44 726 7038.860 46.6427 848 848.511 1.4138 2.49 45 764 6151.769 40.7644 679 1841.145 3.0678 6.40 46 764 6151.769 40.7644 682 1821.592 3.0352 1.04 47 777 5836.727 38.6768 850 837.869 1.3961 2.15 48 777 5836.727 38.6768 853 818.861 1.3644 2.05 49 777 5836.727 38.6768 907 500.256 0.8335 1.64 50 791 5512.022 36.5251 775 1277.340 2.1283 2.38 51 791 5512.022 36.5251 778 1258.929 2.0977 1.38 52 791 5512.022 36.5251 785 1218.892 2.0309 1.38 53 791 5512.022 36.5251 788 1201.847 2.0025 1.09 54 837 4417.038 29.2693 881 655.973 1.0930 23.74 55 848 4174.679 27.6633 864 757.206 1.2617 20.53 56 848 4174.679 27.6633 872 709.060 1.1815 3.17 57 856 3984.149 26.4008 774 1281.339 2.1350 1.66 58 856 3984.149 26.4008 778 1259.751 2.0990 1.59 59 856 3984.149 26.4008 804 1107.062 1.8446 1.02 60 856 3984.149 26.4008 807 1089.219 1.8149 1.21 61 873 3578.068 23.7099 799 1138.137 1.8964 1.68 62 873 3578.068 23.7099 814 1046.185 1.7432 1.26 63 891 3142.837 20.8259 788 1200.655 2.0006 13.95 64 891 3142.837 20.8259 804 1107.046 1.8446 28.24 65 894 3086.147 20.4502 788 1200.275 1.9999 3.08 66 894 3086.147 20.4502 804 1106.803 1.8442 1.17 67 894 3086.147 20.4502 815 1041.758 1.7358 27.19 68 894 3086.147 20.4502 858 789.804 1.3160 1.16 69 897 3015.337 19.9810 858 790.292 1.3168 33.47 70 906 2802.854 18.5730 830 953.652 1.5890 1.35 71 906 2802.854 18.5730 834 931.208 1.5516 1.89 72 914 2613.995 17.3215 892 592.663 0.9875 12.71 73 919 2490.082 16.5004 875 693.445 1.1554 8.90 74 919 2490.082 16.5004 880 660.343 1.1003 1.76 75 925 2342.840 15.5247 778 1258.345 2.0967 5.45 76 925 2342.840 15.5247 782 1237.122 2.0613 5.31 77 925 2342.840 15.5247 791 1183.410 1.9718 2.33 78 925 2342.840 15.5247 795 1159.732 1.9324 2.38 79 925 2342.840 15.5247 907 503.260 0.8385 6.27 80 925 2342.840 15.5247 912 474.699 0.7910 7.15

TABLE 5.3 Chemical Shift Data of 2D NMR chemical shift of HMBC analysis of Y DU = C:/Bruker/XWIN-NMR, USER = guest, NAME = chan, EXPNO = 6, PROCNO = 1 F1PLO = 178.339 ppm, F1PHI = 10.721 ppm, F2PLO = 6.881 ppm, F2PHI = 0.573 ppm MI = 1.00 cm, MAXI = 10000.00 cm, PC = 1.400 ADDRESS FREQUENCY row [Hz]F1 [PPM]F1 # col [Hz]F2 [PPM]F2 INTENSITY 1 123 26590.750 176.2058 895 573.276 0.9552 3.11 2 145 25939.373 171.8894 531 2710.686 4.5166 2.92 3 145 25939.373 171.8894 539 2662.033 4.4355 2.54 4 166 25312.006 167.7321 308 4016.437 6.6923 3.45 5 166 25312.006 167.7321 346 3793.847 6.3214 14.90 6 166 25312.006 167.7321 348 3785.322 6.3072 12.47 7 166 25312.006 167.7321 385 3564.443 5.9392 1.47 8 166 25312.006 167.7321 400 3477.948 5.7950 4.16 9 166 25312.006 167.7321 403 3458.552 5.7627 3.96 10 166 25312.006 167.7321 781 1243.983 2.0728 1.17 11 166 25312.006 167.7321 788 1200.447 2.0002 10.44 12 166 25312.006 167.7321 793 1171.992 1.9528 3.35 13 166 25312.006 167.7321 815 1041.336 1.7351 33.24 14 290 21640.068 143.3997 679 1839.970 3.0658 5.67 15 290 21640.068 143.3997 788 1199.609 1.9988 1.03 16 290 21640.068 143.3997 804 1107.222 1.8449 33.90 17 290 21640.068 143.3997 813 1053.918 1.7561 2.30 18 290 21640.068 143.3997 848 848.155 1.4132 1.25 19 322 20697.354 137.1527 780 1246.505 2.0770 31.46 20 322 20697.354 137.1527 788 1200.075 1.9996 44.60 21 322 20697.354 137.1527 793 1170.377 1.9501 3.79 22 322 20697.354 137.1527 815 1040.860 1.7343 4.31 23 327 20566.367 136.2847 780 1246.629 2.0772 3.06 24 327 20566.367 136.2847 788 1201.192 2.0015 5.78 25 327 20566.367 136.2847 793 1170.748 1.9507 47.25 26 327 20566.367 136.2847 815 1041.067 1.7346 58.19 27 365 19434.006 128.7811 780 1245.861 2.0759 16.02 28 365 19434.006 128.7811 788 1200.442 2.0002 33.73 29 365 19434.006 128.7811 793 1171.204 1.9515 46.70 30 365 19434.006 128.7811 815 1040.940 1.7344 80.60 31 384 18893.113 125.1968 679 1839.547 3.0651 9.80 32 384 18893.113 125.1968 788 1200.729 2.0007 2.34 33 384 18893.113 125.1968 801 1124.942 1.8744 1.93 34 384 18893.113 125.1968 803 1113.960 1.8561 1.36 35 384 18893.113 125.1968 809 1077.556 1.7954 3.32 36 384 18893.113 125.1968 813 1051.694 1.7524 3.14 37 384 18893.113 125.1968 848 847.499 1.4121 1.45 38 457 16738.236 110.9173 361 3707.331 6.1772 6.44 39 457 16738.236 110.9173 390 3534.169 5.8887 6.75 40 457 16738.236 110.9173 486 2972.803 4.9534 1.24 41 457 16738.236 110.9173 488 2962.223 4.9357 1.40 42 457 16738.236 110.9173 563 2520.559 4.1998 12.21 43 488 15822.760 104.8508 531 2712.055 4.5189 1.39 44 488 15822.760 104.8508 538 2668.887 4.4470 4.68 45 488 15822.760 104.8508 545 2627.709 4.3783 14.61 46 488 15822.760 104.8508 660 1952.474 3.2533 2.34 47 488 15822.760 104.8508 662 1941.101 3.2343 3.52 48 488 15822.760 104.8508 664 1928.930 3.2140 2.41 49 489 15777.470 104.5507 538 2669.101 4.4473 12.69 50 489 15777.470 104.5507 546 2622.941 4.3704 6.06 51 489 15777.470 104.5507 590 2363.695 3.9384 3.08 52 489 15777.470 104.5507 660 1953.443 3.2549 2.02 53 489 15777.470 104.5507 662 1940.913 3.2340 1.47 54 566 13527.920 89.6439 492 2938.198 4.8957 11.15 55 566 13527.920 89.6439 805 1101.852 1.8359 1.93 56 566 13527.920 89.6439 851 830.552 1.3839 5.09 57 566 13527.920 89.6439 864 757.374 1.2620 53.64 58 566 13527.920 89.6439 874 696.110 1.1599 39.08 59 566 13527.920 89.6439 880 658.836 1.0978 4.62 60 566 13527.920 89.6439 911 480.432 0.8005 1.38 61 566 13527.920 89.6439 913 466.506 0.7773 1.40 62 584 12992.842 86.0981 376 3621.487 6.0342 5.29 63 584 12992.842 86.0981 531 2708.564 4.5131 1.17 64 584 12992.842 86.0981 539 2662.648 4.4366 4.18 65 584 12992.842 86.0981 545 2625.947 4.3754 6.92 66 584 12992.842 86.0981 864 757.040 1.2614 1.36 67 588 12860.941 85.2241 370 3655.044 6.0901 2.76 68 588 12860.941 85.2241 376 3621.609 6.0344 10.88 69 588 12860.941 85.2241 554 2574.294 4.2893 1.21 70 588 12860.941 85.2241 864 756.227 1.2600 1.05 71 598 12585.614 83.3996 376 3621.268 6.0338 1.92 72 598 12585.614 83.3996 475 3038.901 5.0635 1.23 73 598 12585.614 83.3996 500 2890.984 4.8170 1.60 74 598 12585.614 83.3996 505 2863.233 4.7708 3.39 75 598 12585.614 83.3996 881 655.156 1.0916 1.11 76 623 11822.784 78.3447 295 4093.818 6.8212 2.30 77 623 11822.784 78.3447 321 3940.803 6.5663 2.25 78 623 11822.784 78.3447 348 3785.120 6.3069 18.28 79 623 11822.784 78.3447 434 3277.040 5.4603 1.13 80 623 11822.784 78.3447 448 3194.918 5.3234 7.26 81 623 11822.784 78.3447 563 2521.171 4.2008 7.36 82 623 11822.784 78.3447 845 867.271 1.4451 1.26 83 623 11822.784 78.3447 849 844.669 1.4074 10.09 84 623 11822.784 78.3447 858 790.448 1.3171 26.63 85 623 11822.784 78.3447 881 656.434 1.0938 51.16 86 630 11619.674 76.9987 348 3785.085 6.3068 1.21 87 630 11619.674 76.9987 539 2661.936 4.4354 6.49 88 630 11619.674 76.9987 552 2584.607 4.3065 1.49 89 630 11619.674 76.9987 858 790.978 1.3179 2.74 90 630 11619.674 76.9987 881 655.977 1.0930 5.21 91 641 11304.499 74.9102 307 4021.328 6.7004 1.03 92 641 11304.499 74.9102 526 2736.013 4.5588 4.14 93 641 11304.499 74.9102 538 2670.608 4.4498 9.80 94 641 11304.499 74.9102 881 656.537 1.0939 1.61 95 650 11028.925 73.0841 308 4015.948 6.6915 16.79 96 650 11028.925 73.0841 346 3793.459 6.3207 13.94 97 650 11028.925 73.0841 526 2736.761 4.5601 7.46 98 650 11028.925 73.0841 574 2454.410 4.0896 4.70 99 650 11028.925 73.0841 610 2245.856 3.7421 11.08 100 650 11028.925 73.0841 637 2088.895 3.4806 5.03 101 650 11028.925 73.0841 679 1839.510 3.0650 8.83 102 650 11028.925 73.0841 848 850.226 1.4167 1.52 103 650 11028.925 73.0841 881 656.986 1.0947 1.80 104 658 10790.329 71.5030 308 4016.477 6.6923 1.26 105 658 10790.329 71.5030 531 2708.840 4.5135 2.58 106 658 10790.329 71.5030 564 2518.203 4.1959 5.49 107 658 10790.329 71.5030 609 2253.841 3.7554 1.07 108 658 10790.329 71.5030 881 655.677 1.0925 1.26 109 668 10496.015 69.5527 530 2715.370 4.5244 1.73 110 668 10496.015 69.5527 533 2699.760 4.4984 1.85 111 668 10496.015 69.5527 540 2658.708 4.4300 3.19 112 668 10496.015 69.5527 590 2363.392 3.9379 4.52 113 668 10496.015 69.5527 804 1107.602 1.8455 1.00 114 668 10496.015 69.5527 881 656.508 1.0939 1.01 115 680 10155.364 67.2954 541 2651.054 4.4172 2.15 116 680 10155.364 67.2954 804 1107.228 1.8449 32.23 117 680 10155.364 67.2954 881 656.545 1.0939 1.00 118 703 9486.470 62.8629 346 3793.169 6.3203 9.40 119 703 9486.470 62.8629 348 3784.896 6.3065 9.63 120 703 9486.470 62.8629 679 1839.346 3.0648 3.08 121 703 9486.470 62.8629 863 758.265 1.2634 1.07 122 706 9376.976 62.1373 347 3790.748 6.3162 1.61 123 706 9376.976 62.1373 505 2862.456 4.7695 2.79 124 706 9376.976 62.1373 881 655.399 1.0920 1.01 125 709 9296.042 61.6010 590 2363.684 3.9384 7.53 126 741 8346.307 55.3075 777 1264.802 2.1074 3.77 127 741 8346.307 55.3075 852 825.908 1.3761 3.30 128 741 8346.307 55.3075 864 757.401 1.2620 35.15 129 741 8346.307 55.3075 874 696.341 1.1603 25.71 130 741 8346.307 55.3075 881 658.199 1.0967 3.92 131 741 8346.307 55.3075 891 595.647 0.9925 1.15 132 741 8346.307 55.3075 909 489.518 0.8156 23.33 133 781 7163.191 47.4675 346 3793.181 6.3203 1.69 134 781 7163.191 47.4675 431 3294.477 5.4893 5.30 135 781 7163.191 47.4675 539 2661.476 4.4346 3.10 136 781 7163.191 47.4675 541 2647.966 4.4121 3.42 137 781 7163.191 47.4675 564 2517.984 4.1955 1.14 138 781 7163.191 47.4675 679 1839.442 3.0649 3.64 139 781 7163.191 47.4675 804 1107.164 1.8448 37.94 140 781 7163.191 47.4675 822 1002.093 1.6697 1.62 141 781 7163.191 47.4675 849 845.058 1.4081 2.98 142 781 7163.191 47.4675 858 790.203 1.3167 4.12 143 781 7163.191 47.4675 874 695.441 1.1588 1.07 144 781 7163.191 47.4675 881 656.256 1.0935 6.01 145 781 7163.191 47.4675 891 594.695 0.9909 39.60 146 781 7163.191 47.4675 909 489.613 0.8158 3.90 147 786 7041.051 46.6581 432 3290.762 5.4831 2.39 148 786 7041.051 46.6581 669 1898.191 3.1628 1.21 149 786 7041.051 46.6581 677 1850.653 3.0836 2.37 150 786 7041.051 46.6581 680 1835.948 3.0591 2.84 151 786 7041.051 46.6581 682 1821.518 3.0351 2.66 152 786 7041.051 46.6581 689 1782.432 2.9699 2.02 153 786 7041.051 46.6581 776 1274.214 2.1231 2.05 154 786 7041.051 46.6581 800 1129.576 1.8821 1.51 155 786 7041.051 46.6581 804 1106.939 1.8444 6.33 156 786 7041.051 46.6581 808 1082.787 1.8042 3.27 157 786 7041.051 46.6581 811 1065.178 1.7748 3.51 158 786 7041.051 46.6581 829 959.508 1.5988 3.10 159 786 7041.051 46.6581 838 906.837 1.5110 1.42 160 786 7041.051 46.6581 848 847.280 1.4118 1.15 161 786 7041.051 46.6581 858 790.530 1.3172 49.82 162 786 7041.051 46.6581 874 696.770 1.1610 1.14 163 786 7041.051 46.6581 881 656.443 1.0938 70.13 164 786 7041.051 46.6581 891 594.532 0.9906 18.99 165 786 7041.051 46.6581 909 489.363 0.8154 21.10 166 814 6218.377 41.2066 431 3293.885 5.4883 1.45 167 814 6218.377 41.2066 561 2531.574 4.2182 3.03 168 814 6218.377 41.2066 564 2517.867 4.1953 3.26 169 814 6218.377 41.2066 635 2096.944 3.4940 2.64 170 814 6218.377 41.2066 678 1846.079 3.0760 2.51 171 814 6218.377 41.2066 776 1274.484 2.1236 1.75 172 814 6218.377 41.2066 783 1228.144 2.0464 1.41 173 814 6218.377 41.2066 786 1211.726 2.0190 1.51 174 814 6218.377 41.2066 799 1136.142 1.8931 1.43 175 814 6218.377 41.2066 804 1107.204 1.8448 67.13 176 814 6218.377 41.2066 819 1018.301 1.6967 4.37 177 814 6218.377 41.2066 822 1001.489 1.6687 4.96 178 814 6218.377 41.2066 833 939.695 1.5657 3.66 179 814 6218.377 41.2066 864 756.271 1.2601 2.02 180 814 6218.377 41.2066 874 695.963 1.1596 2.98 181 814 6218.377 41.2066 880 659.072 1.0982 1.66 182 814 6218.377 41.2066 891 594.287 0.9902 49.75 183 814 6218.377 41.2066 895 572.724 0.9543 2.16 184 814 6218.377 41.2066 909 489.811 0.8161 1.62 185 823 5950.421 39.4310 662 1940.805 3.2338 1.07 186 823 5950.421 39.4310 776 1271.011 2.1178 1.62 187 823 5950.421 39.4310 804 1107.587 1.8455 2.29 188 823 5950.421 39.4310 831 947.726 1.5791 1.04 189 823 5950.421 39.4310 858 790.520 1.3172 1.45 190 823 5950.421 39.4310 864 757.090 1.2615 54.14 191 823 5950.421 39.4310 874 696.048 1.1598 72.55 192 823 5950.421 39.4310 880 658.503 1.0972 9.62 193 823 5950.421 39.4310 891 594.530 0.9906 2.75 194 823 5950.421 39.4310 911 479.432 0.7988 7.32 195 823 5950.421 39.4310 913 465.776 0.7761 7.03 196 827 5834.797 38.6648 804 1106.687 1.8440 2.69 197 827 5834.797 38.6648 819 1017.803 1.6959 1.66 198 827 5834.797 38.6648 822 1001.708 1.6691 1.68 199 827 5834.797 38.6648 841 889.985 1.4829 1.01 200 827 5834.797 38.6648 858 789.621 1.3157 1.33 201 827 5834.797 38.6648 864 758.164 1.2633 3.94 202 827 5834.797 38.6648 874 696.137 1.1599 6.09 203 827 5834.797 38.6648 881 656.905 1.0945 2.43 204 827 5834.797 38.6648 892 593.606 0.9891 1.90 205 827 5834.797 38.6648 909 489.245 0.8152 51.13 206 830 5743.329 38.0586 804 1108.031 1.8462 1.40 207 830 5743.329 38.0586 858 790.551 1.3172 1.95 208 830 5743.329 38.0586 864 757.091 1.2615 2.85 209 830 5743.329 38.0586 874 696.052 1.1598 3.54 210 830 5743.329 38.0586 881 655.620 1.0924 2.41 211 830 5743.329 38.0586 891 594.762 0.9910 3.06 212 830 5743.329 38.0586 909 489.474 0.8156 10.76 213 834 5623.896 37.2672 804 1107.433 1.8452 1.25 214 834 5623.896 37.2672 822 1002.039 1.6696 1.43 215 834 5623.896 37.2672 833 939.620 1.5656 1.01 216 834 5623.896 37.2672 858 790.603 1.3173 4.03 217 834 5623.896 37.2672 864 756.768 1.2609 1.89 218 834 5623.896 37.2672 874 696.444 1.1604 2.15 219 834 5623.896 37.2672 881 656.011 1.0931 4.39 220 834 5623.896 37.2672 891 594.776 0.9910 4.19 221 834 5623.896 37.2672 909 489.272 0.8152 10.53 222 837 5533.835 36.6704 777 1268.681 2.1139 1.72 223 837 5533.835 36.6704 804 1108.282 1.8466 1.01 224 837 5533.835 36.6704 811 1067.120 1.7781 1.70 225 837 5533.835 36.6704 819 1017.407 1.6952 4.37 226 837 5533.835 36.6704 822 1001.730 1.6691 4.67 227 837 5533.835 36.6704 832 943.762 1.5725 2.78 228 837 5533.835 36.6704 849 841.934 1.4028 2.09 229 837 5533.835 36.6704 858 790.630 1.3174 8.57 230 837 5533.835 36.6704 864 757.963 1.2629 2.10 231 837 5533.835 36.6704 874 695.245 1.1584 1.83 232 837 5533.835 36.6704 881 656.351 1.0936 11.03 233 837 5533.835 36.6704 891 594.692 0.9909 25.74 234 837 5533.835 36.6704 909 489.139 0.8150 53.53 235 837 5533.835 36.6704 913 467.893 0.7796 6.55 236 840 5450.419 36.1177 307 4023.448 6.7040 5.35 237 840 5450.419 36.1177 309 4013.866 6.6880 5.05 238 840 5450.419 36.1177 346 3794.546 6.3226 1.25 239 840 5450.419 36.1177 679 1839.242 3.0646 3.88 240 840 5450.419 36.1177 849 842.830 1.4043 6.34 241 840 5450.419 36.1177 858 790.237 1.3167 88.69 242 840 5450.419 36.1177 862 767.125 1.2782 1.79 243 840 5450.419 36.1177 864 755.041 1.2581 1.99 244 840 5450.419 36.1177 874 695.441 1.1588 2.24 245 840 5450.419 36.1177 881 656.060 1.0931 84.89 246 840 5450.419 36.1177 891 594.675 0.9909 7.64 247 840 5450.419 36.1177 909 489.300 0.8153 8.88 248 875 4418.238 29.2778 307 4022.778 6.7028 8.21 249 875 4418.238 29.2778 309 4014.385 6.6889 7.83 250 875 4418.238 29.2778 679 1841.576 3.0685 1.85 251 875 4418.238 29.2778 849 844.669 1.4074 1.65 252 875 4418.238 29.2778 858 790.492 1.3171 100.00 253 875 4418.238 29.2778 863 763.811 1.2727 3.72 254 875 4418.238 29.2778 870 718.885 1.1978 22.51 255 875 4418.238 29.2778 875 691.903 1.1529 4.90 256 875 4418.238 29.2778 878 670.147 1.1166 1.92 257 875 4418.238 29.2778 882 648.470 1.0805 1.77 258 875 4418.238 29.2778 892 593.301 0.9886 19.66 259 883 4176.082 27.6732 662 1941.300 3.2346 1.71 260 883 4176.082 27.6732 853 819.336 1.3652 15.64 261 883 4176.082 27.6732 857 794.801 1.3243 4.59 262 883 4176.082 27.6732 863 763.374 1.2720 1.70 263 883 4176.082 27.6732 874 696.058 1.1598 89.76 264 883 4176.082 27.6732 878 671.861 1.1195 2.96 265 883 4176.082 27.6732 881 657.198 1.0950 7.96 266 883 4176.082 27.6732 911 479.487 0.7989 2.54 267 883 4176.082 27.6732 913 466.645 0.7775 2.27 268 903 3588.775 23.7813 431 3296.117 5.4921 2.85 269 903 3588.775 23.7813 791 1182.756 1.9707 1.05 270 903 3588.775 23.7813 801 1127.886 1.8793 1.45 271 903 3588.775 23.7813 804 1108.540 1.8471 1.43 272 903 3588.775 23.7813 811 1064.128 1.7731 1.37 273 903 3588.775 23.7813 821 1006.100 1.6764 3.82 274 903 3588.775 23.7813 874 696.649 1.1608 2.42 275 903 3588.775 23.7813 881 655.757 1.0926 2.43 276 917 3174.632 21.0370 307 4021.419 6.7006 1.44 277 917 3174.632 21.0370 384 3571.435 5.9508 2.36 278 917 3174.632 21.0370 403 3463.331 5.7707 1.11 279 917 3174.632 21.0370 561 2531.850 4.2186 5.21 280 917 3174.632 21.0370 564 2517.700 4.1950 5.22 281 917 3174.632 21.0370 778 1262.748 2.1040 3.52 282 917 3174.632 21.0370 793 1170.055 1.9496 22.51 283 917 3174.632 21.0370 800 1133.275 1.8883 2.77 284 917 3174.632 21.0370 805 1100.419 1.8335 3.08 285 917 3174.632 21.0370 809 1079.427 1.7986 2.91 286 917 3174.632 21.0370 815 1043.118 1.7381 24.13 287 917 3174.632 21.0370 827 975.020 1.6246 2.05 288 917 3174.632 21.0370 848 851.242 1.4184 1.60 289 917 3174.632 21.0370 864 757.267 1.2618 1.67 290 917 3174.632 21.0370 869 727.306 1.2119 1.80 291 917 3174.632 21.0370 881 656.407 1.0937 6.11 292 922 3013.427 19.9687 309 4014.743 6.6895 13.55 293 922 3013.427 19.9687 401 3472.374 5.7857 2.11 294 922 3013.427 19.9687 679 1841.875 3.0690 7.26 295 922 3013.427 19.9687 777 1263.952 2.1060 1.96 296 922 3013.427 19.9687 794 1165.725 1.9424 1.57 297 922 3013.427 19.9687 799 1137.940 1.8961 2.13 298 922 3013.427 19.9687 804 1104.456 1.8403 3.32 299 922 3013.427 19.9687 815 1043.070 1.7380 1.92 300 922 3013.427 19.9687 826 975.688 1.6257 3.29 301 922 3013.427 19.9687 847 852.676 1.4207 19.79 302 922 3013.427 19.9687 863 759.553 1.2656 3.39 303 922 3013.427 19.9687 869 726.647 1.2108 19.23 304 922 3013.427 19.9687 881 656.158 1.0933 72.09 305 929 2803.190 18.5756 308 4021.188 6.7002 1.12 306 929 2803.190 18.5756 823 997.155 1.6615 1.03 307 929 2803.190 18.5756 847 852.409 1.4203 2.46 308 929 2803.190 18.5756 864 756.417 1.2604 2.78 309 929 2803.190 18.5756 869 728.727 1.2142 2.15 310 929 2803.190 18.5756 881 657.345 1.0953 5.08 311 929 2803.190 18.5756 902 531.583 0.8857 1.25 312 929 2803.190 18.5756 911 478.022 0.7965 4.88 313 929 2803.190 18.5756 913 466.494 0.7773 5.42 314 936 2611.793 17.3073 777 1266.005 2.1094 1.92 315 936 2611.793 17.3073 783 1228.491 2.0469 2.67 316 936 2611.793 17.3073 786 1211.785 2.0191 2.98 317 936 2611.793 17.3073 819 1018.301 1.6967 5.90 318 936 2611.793 17.3073 822 1001.860 1.6693 7.01 319 936 2611.793 17.3073 864 757.418 1.2620 6.44 320 936 2611.793 17.3073 869 725.753 1.2093 1.48 321 936 2611.793 17.3073 872 707.264 1.1785 1.56 322 936 2611.793 17.3073 881 654.782 1.0910 17.83 323 936 2611.793 17.3073 902 531.793 0.8861 19.86 324 936 2611.793 17.3073 909 490.194 0.8168 2.94 325 936 2611.793 17.3073 920 427.649 0.7126 1.52 326 940 2489.586 16.4974 662 1941.819 3.2355 2.46 327 940 2489.586 16.4974 664 1929.172 3.2144 1.76 328 940 2489.586 16.4974 822 1001.010 1.6679 1.20 329 940 2489.586 16.4974 864 757.021 1.2614 66.00 330 940 2489.586 16.4974 871 712.622 1.1874 10.06 331 940 2489.586 16.4974 880 660.618 1.1007 2.75 332 940 2489.586 16.4974 885 632.387 1.0537 18.76 333 940 2489.586 16.4974 891 594.244 0.9901 3.19 334 940 2489.586 16.4974 899 548.142 0.9133 1.83 335 940 2489.586 16.4974 911 478.615 0.7975 8.03 336 940 2489.586 16.4974 913 466.429 0.7772 10.10 337 940 2489.586 16.4974 920 426.462 0.7106 1.57 338 945 2348.374 15.5617 383 3576.612 5.9594 2.25 339 945 2348.374 15.5617 402 3464.839 5.7732 1.77 340 945 2348.374 15.5617 769 1311.156 2.1847 9.92 341 945 2348.374 15.5617 783 1233.208 2.0548 4.90 342 945 2348.374 15.5617 791 1184.039 1.9729 8.41 343 945 2348.374 15.5617 804 1106.429 1.8436 5.06 344 945 2348.374 15.5617 819 1018.365 1.6968 3.91 345 945 2348.374 15.5617 822 1001.834 1.6693 5.15 346 945 2348.374 15.5617 864 756.741 1.2609 6.60 347 945 2348.374 15.5617 872 710.417 1.1837 1.20 348 945 2348.374 15.5617 881 655.039 1.0914 2.79 349 945 2348.374 15.5617 885 632.486 1.0539 2.24 350 945 2348.374 15.5617 899 550.147 0.9167 19.61 351 945 2348.374 15.5617 906 510.518 0.8506 3.78 352 945 2348.374 15.5617 911 480.301 0.8003 8.74 353 945 2348.374 15.5617 914 464.362 0.7737 8.49 354 945 2348.374 15.5617 920 426.538 0.7107 18.76

TABLE 5.5 Chemical Shift of the Proton NMR of compound Y of Xanthoceras Sorbifolia Extract FREQUENCY # ADDRESS [Hz] [PPM] INTENSITY 1 5031.9 5309.524 8.8468 0.46 2 5430.0 5222.264 8.7015 134.66 3 5840.9 5132.173 8.5513 0.48 4 8204.9 4613.901 7.6878 0.26 5 8568.1 4534.263 7.5551 88.28 6 8943.7 4451.919 7.4179 0.33 7 9209.7 4393.602 7.3207 0.80 8 9575.2 4313.488 7.1872 180.00 9 9952.1 4230.846 7.0495 0.87 10 10277.2 4159.585 6.9308 1.02 11 10886.8 4025.922 6.7081 5.68 12 10933.6 4015.677 6.6910 6.19 13 11939.8 3795.084 6.3235 6.26 14 11986.7 3784.803 6.3063 5.85 15 12576.0 3655.587 6.0910 1.47 16 12728.0 3622.280 6.0355 6.92 17 12880.5 3588.844 5.9798 1.87 18 12914.2 3581.450 5.9675 4.48 19 12946.5 3574.366 5.9557 4.59 20 12979.5 3567.130 5.9436 2.00 21 13382.6 3478.754 5.7964 2.41 22 13415.6 3471.539 5.7844 5.03 23 13447.3 3464.568 5.7727 5.15 24 13479.1 3457.598 5.7611 2.66 25 14218.8 3295.432 5.4909 14.00 26 14655.9 3199.603 5.3312 28.97 27 14691.2 3191.875 5.3184 27.63 28 15715.3 2967.359 4.9443 5.64 29 15826.3 2943.028 4.9037 4.82 30 15860.6 2935.504 4.8912 5.15 31 16047.0 2894.632 4.8231 4.20 32 16149.6 2872.131 4.7856 3.79 33 16171.9 2867.242 4.7775 4.42 34 16774.0 2735.249 4.5575 5.92 35 16856.4 2717.187 4.5274 4.26 36 16883.5 2711.235 4.5175 4.56 37 16903.5 2706.854 4.5102 5.89 38 16930.6 2700.914 4.5003 4.68 39 17021.2 2681.046 4.4672 2.99 40 17056.9 2673.228 4.4542 8.41 41 17099.1 2663.974 4.4388 8.87 42 17135.6 2655.976 4.4254 8.11 43 17200.4 2641.771 4.4018 1.88 44 17232.6 2634.706 4.3900 2.90 45 17271.0 2626.288 4.3760 4.33 46 17308.8 2618.001 4.3622 2.85 47 17401.8 2597.607 4.3282 0.83 48 17459.1 2585.043 4.3073 3.41 49 17512.6 2573.328 4.2877 4.04 50 17596.4 2554.947 4.2571 0.89 51 17628.2 2547.968 4.2455 0.80 52 17666.8 2539.518 4.2314 1.30 53 17723.0 2527.184 4.2109 6.71 54 17745.9 2522.172 4.2025 7.98 55 17791.2 2512.248 4.1860 2.28 56 17874.1 2494.071 4.1557 2.90 57 17895.0 2489.477 4.1480 3.09 58 17927.1 2482.454 4.1363 2.64 59 17948.8 2477.683 4.1284 2.51 60 18034.1 2458.993 4.0972 3.27 61 18064.6 2452.313 4.0861 2.96 62 18076.5 2449.702 4.0817 2.96 63 18118.3 2440.538 4.0665 0.72 64 18156.4 2432.180 4.0526 0.89 65 18196.3 2423.432 4.0380 0.44 66 18432.1 2371.735 3.9518 2.34 67 18459.7 2365.684 3.9418 4.30 68 18487.4 2359.600 3.9316 2.24 69 18962.4 2255.466 3.7581 3.86 70 19011.0 2244.816 3.7404 4.38 71 19669.3 2100.485 3.4999 4.39 72 19717.6 2089.898 3.4822 4.16 73 20365.2 1947.920 3.2457 2.42 74 20401.1 1940.054 3.2326 2.35 75 20418.9 1936.161 3.2261 2.38 76 20751.3 1863.290 3.1047 0.70 77 20815.8 1849.152 3.0811 3.20 78 20857.9 1839.919 3.0657 11.24 79 20924.8 1825.236 3.0412 1.13 80 23431.3 1275.730 2.1257 3.95 81 23491.3 1262.573 2.1037 5.36 82 23538.7 1252.187 2.0864 15.56 83 23570.5 1245.203 2.0748 15.89 84 23696.1 1217.673 2.0289 2.98 85 23770.1 1201.463 2.0019 27.17 86 23827.6 1188.849 1.9809 1.36 87 23887.4 1175.734 1.9590 15.22 88 23919.5 1168.700 1.9473 15.28 89 24010.2 1148.811 1.9142 0.49 90 24104.2 1128.207 1.8798 1.77 91 24197.7 1107.716 1.8457 28.22 92 24263.8 1093.228 1.8216 3.27 93 24321.8 1080.503 1.8004 2.93 94 24373.5 1069.166 1.7815 2.83 95 24497.0 1042.094 1.7364 26.10 96 24598.4 1019.871 1.6993 3.17 97 24626.1 1013.793 1.6892 3.14 98 24676.1 1002.826 1.6709 1.84 99 24931.2 946.900 1.5777 2.14 100 24983.0 935.533 1.5588 3.02 101 25226.8 882.103 1.4698 0.25 102 25370.4 850.608 1.4173 4.11 103 25412.0 841.492 1.4021 4.88 104 25499.7 822.270 1.3701 4.07 105 25556.8 809.746 1.3492 2.62 106 25639.9 791.527 1.3189 31.95 107 25717.9 774.418 1.2904 2.84 108 25790.4 758.539 1.2639 22.85 109 26011.7 710.018 1.1830 4.66 110 26070.7 697.082 1.1615 24.95 111 26249.2 657.953 1.0963 31.39 112 26536.4 594.981 0.9914 25.26 113 26610.9 578.657 0.9642 0.97 114 26914.0 512.196 0.8534 2.04 115 27012.2 490.676 0.8176 25.88 116 27118.1 467.463 0.7789 3.69 117 27226.4 443.715 0.7393 1.07 118 28513.4 161.554 0.2692 0.89 119 28539.8 155.777 0.2596 1.54

TABLE 6.2 HMQC of Y1 peaklist # # ADDRESS FREQUENCY INTENSITY 1 1 315 16740.193 110.9281 30.72 375 3624.445 6.0391 2 352 15855.943 105.0686 20.34 490 2950.420 4.9161 3 450 13537.575 89.7061 20.34 490 2950.420 4.9161 4 479 12863.844 85.2416 12.42 476 3032.063 5.0521 5 479 12863.844 85.2416 17.76 500 2892.360 4.8193 6 491 12577.806 83.3462 25.72 487 2969.087 4.9472 7 522 11857.070 78.5703 13.55 545 2627.024 4.3772 8 529 11685.553 77.4337 16.93 504 2870.087 4.7822 9 535 11537.104 76.4500 15.06 589 2370.150 3.9492 10 550 11191.633 74.1608 12.94 421 3355.320 5.5907 11 550 11191.633 74.1608 15.14 423 3342.807 5.5699 12 569 10733.316 71.1238 24.93 410 3421.295 5.7006 13 579 10497.793 69.5631 26.50 526 2740.480 4.5662 14 586 10332.951 68.4708 14.53 537 2674.056 4.4556 15 586 10332.951 68.4708 11.00 596 2330.159 3.8826 16 617 9621.364 63.7555 12.23 643 2051.886 3.4189 17 630 9299.908 61.6254 13.25 529 2718.381 4.5294 18 630 9299.908 61.6254 14.42 539 2665.365 4.4411 19 669 8391.979 55.6090 13.25 529 2718.381 4.5294 20 669 8391.979 55.6090 14.42 539 2665.365 4.4411 21 720 7193.972 47.6705 10.83 682 1821.991 3.0358 22 725 7055.427 46.7524 8.84 820 1015.280 1.6917 23 770 5994.782 39.7241 21.78 682 1822.241 3.0363 24 778 5810.328 38.5019 21.78 682 1822.241 3.0363 25 803 5229.784 34.6549 9.20 836 918.974 1.5312 26 813 4978.483 32.9897 9.04 866 743.550 1.2389 27 833 4517.225 29.9332 64.24 847 852.860 1.4211 28 847 4185.850 27.7373 74.68 861 773.417 1.2887 29 850 4113.891 27.2605 80.21 806 1096.582 1.8271 30 850 4113.891 27.2605 31.46 861 773.392 1.2886 31 885 3291.884 21.8135 100.00 756 1389.088 2.3145 32 893 3110.641 20.6125 89.09 796 1153.093 1.9213 33 893 3110.641 20.6125 50.66 803 1110.425 1.8502 34 897 2998.505 19.8694 45.64 803 1111.101 1.8513 35 897 2998.505 19.8694 78.07 853 820.195 1.3666 36 919 2497.783 16.5514 72.04 874 699.217 1.1651 37 919 2497.783 16.5514 37.18 910 483.491 0.8056 38 926 2336.725 15.4842 17.70 794 1165.059 1.9412 39 926 2336.725 15.4842 31.53 910 484.392 0.8071 row [Hz]F1 [PPM]F1 col [Hz]F2 [PPM]F2

TABLE 6.2A 2D NMR (HMQC) data of Y1 Assignment w1 w2 Data Height  1C-H 110.904 6.038 24704052  2C-H 105.023 4.914 15964054  3C-H 104.615 5.339 11824459  4C-H 89.823 3.243 7951700  5C-H 89.712 3.222 7911944  6C-H 85.528 5.038 10494958  7C-H 85.417 5.054 8839478  8C-H 85.338 4.831 10297378  9C-H 85.248 4.817 12857784 10C-H 83.397 4.948 19288902 11C-H 78.655 4.381 7900396 12C-H 78.602 4.366 8972991 13C-H 77.420 4.784 13438428 14C-H 77.007 4.497 7206874 15C-H 76.570 3.951 12216028 16C-H 74.994 4.092 11486882 17C-H 74.790 4.106 12904558 18C-H 74.220 5.593 10130728 19C-H 74.062 5.574 9532875 20C-H 73.856 6.173 11098625 21C-H 73.802 6.156 10393206 22C-H 73.350 4.468 11007188 23C-H 73.277 4.446 7281630 24C-H 71.577 4.453 8645994 25C-H 71.219 5.701 24595648 26C-H 70.611 3.615 9076031 27C-H 70.067 4.256 9261139 28C-H 69.616 4.567 18994736 29C-H 68.771 3.895 8451744 30C-H 68.543 4.455 12573076 31C-H 63.679 3.604 8239119 32C-H 63.781 3.415 7458621 33C-H 63.862 3.393 7841054 34C-H 62.192 4.312 10795595 35C-H 62.172 4.287 8408334 36C-H 62.266 4.162 7944312 37C-H 62.071 4.131 10031945 38C-H 61.811 4.529 12476046 39C-H 61.793 4.440 11880108 40C-H 61.705 4.463 8055856 41C-H 47.663 3.034 7772454 42C-H 46.740 1.693 8583785 43C-H 39.795 3.034 18038864 44C-H 38.453 1.337 7912356 45C-H 34.666 1.567 7317314 46C-H 32.884 1.264 7887187 47C-H 29.954 1.421 58253544 48C-H 29.632 1.242 16326471 49C-H 29.318 1.117 9248448 50C-H 27.723 1.292 47380240 51C-H 27.251 1.831 59260700 52C-H 21.835 2.317 68135008 53C-H 20.604 1.919 59543524 54C-H 20.377 1.852 66987324 55C-H 19.850 1.369 68067232 56C-H 16.547 1.167 54857956 57C-H 16.223 0.807 25159514 58C-H 15.890 2.028 14256620 59C-H 15.674 1.978 12750140 60C-H 15.693 1.940 10562185 61C-H 15.378 0.758 16911768 62C-H 15.149 0.799 12756197 63C-H 138.670 5.985 8925989 64C-H 137.837 5.881 6871625 65C-H 123.420 5.365 15877691

TABLE 6.3 2D NMR (HMBC) data of Y1 peaklist # ADDRESS FREQUENCY INTENSITY 1 148 25871.928 171.4391 11.03 362 3702.490 6.1692 2 148 25871.928 171.4391 93.44 756 1390.051 2.3161 3 169 25232.139 167.1995 28.04 796 1153.459 1.9219 4 169 25232.139 167.1995 29.81 803 1112.088 1.8530 5 293 21555.236 142.8347 25.74 806 1097.351 1.8284 6 314 20935.686 138.7293 33.12 786 1215.627 2.0255 7 314 20935.686 138.7293 46.88 796 1153.008 1.9212 8 318 20813.068 137.9168 9.66 786 1213.951 2.0227 9 318 20813.068 137.9168 29.47 792 1175.438 1.9585 10 318 20813.068 137.9168 46.19 803 1110.584 1.8505 11 369 19321.684 128.0342 13.34 785 1218.100 2.0296 12 369 19321.684 128.0342 28.29 792 1175.050 1.9579 13 369 19321.684 128.0342 26.99 796 1153.691 1.9223 14 369 19321.684 128.0342 65.83 803 1111.953 1.8528 15 486 15874.223 105.1898 16.48 477 3026.433 5.0427 16 486 15874.223 105.1898 9.96 545 2628.184 4.3791 17 486 15874.223 105.1898 10.46 557 2559.578 4.2648 18 565 13533.447 89.6787 10.78 491 2942.427 4.9027 19 565 13533.447 89.6787 41.26 861 774.469 1.2904 20 565 13533.447 89.6787 36.06 874 696.834 1.1611 21 587 12903.256 85.5028 20.78 362 3702.844 6.1698 22 587 12903.256 85.5028 12.97 375 3625.743 6.0413 23 587 12903.256 85.5028 35.15 847 852.970 1.4212 24 587 12903.256 85.5028 30.07 853 821.094 1.3681 25 622 11874.164 78.6836 9.13 447 3202.899 5.3367 26 622 11874.164 78.6836 9.75 563 2519.490 4.1980 27 647 11136.594 73.7961 19.44 476 3029.853 5.0484 28 647 11136.594 73.7961 9.88 625 2159.425 3.5981 29 660 10736.229 71.1431 30.15 873 702.447 1.1704 30 668 10508.083 69.6313 11.82 873 702.134 1.1699 31 673 10374.060 68.7432 41.29 873 702.539 1.1706 32 709 9310.579 61.6961 9.48 588 2374.061 3.9557 33 740 8380.918 55.5357 42.71 861 775.074 1.2914 34 740 8380.918 55.5357 30.33 874 696.643 1.1608 35 740 8380.918 55.5357 21.32 913 469.072 0.7816 36 781 7183.864 47.6035 43.72 847 853.277 1.4217 37 781 7183.864 47.6035 35.29 853 821.688 1.3691 38 786 7044.034 46.6769 9.09 848 851.746 1.4192 39 786 7044.034 46.6769 8.36 880 661.983 1.1030 40 786 7044.034 46.6769 27.00 913 469.396 0.7821 41 813 6236.901 41.3285 38.52 806 1097.285 1.8283 42 813 6236.901 41.3285 43.99 910 485.026 0.8082 43 822 5962.607 39.5109 32.36 806 1097.624 1.8289 44 822 5962.607 39.5109 41.54 861 774.567 1.2906 45 822 5962.607 39.5109 63.83 874 696.338 1.1603 46 822 5962.607 39.5109 32.10 910 484.561 0.8074 47 828 5792.666 38.3848 9.58 853 821.024 1.3680 48 828 5792.666 38.3848 39.07 913 469.203 0.7818 49 832 5676.145 37.6127 42.80 847 852.643 1.4207 50 832 5676.145 37.6127 53.76 853 821.900 1.3695 51 832 5676.145 37.6127 10.57 912 471.157 0.7851 52 837 5531.661 36.6553 8.78 853 822.175 1.3699 53 837 5531.661 36.6553 45.56 913 468.520 0.7807 54 847 5226.124 34.6307 27.35 805 1099.040 1.8312 55 856 4961.126 32.8747 32.47 910 484.632 0.8075 56 871 4515.250 29.9201 7.66 477 3025.946 5.0419 57 871 4515.250 29.9201 8.91 837 913.407 1.5219 58 871 4515.250 29.9201 52.28 853 821.483 1.3688 59 883 4184.067 27.7255 8.46 795 1160.819 1.9342 60 883 4184.067 27.7255 11.68 850 836.992 1.3946 61 883 4184.067 27.7255 77.80 874 696.591 1.1607 62 913 3294.604 21.8315 16.90 745 1453.362 2.4216 63 913 3294.604 21.8315 15.41 767 1324.146 2.2063 64 923 2998.099 19.8668 12.57 477 3025.408 5.0410 65 923 2998.099 19.8668 9.99 683 1819.240 3.0313 66 923 2998.099 19.8668 18.02 842 883.190 1.4716 67 923 2998.099 19.8668 51.49 847 853.156 1.4215 68 923 2998.099 19.8668 16.19 864 757.915 1.2629 69 923 2998.099 19.8668 10.30 879 666.328 1.1103 70 940 2480.249 16.4352 42.66 861 775.278 1.2918 71 940 2480.249 16.4352 14.60 884 636.584 1.0607 72 940 2480.249 16.4352 12.91 900 544.736 0.9077 73 940 2480.249 16.4352 12.28 921 420.806 0.7012 74 946 2313.568 15.3307 14.80 902 531.201 0.8851 75 946 2313.568 15.3307 12.79 923 407.732 0.6794 row [Hz]F1 [PPM]F1 col [Hz]F2 [PPM]F2

TABLE 6.4. 2D NMR (COSY) data of Y1. Assignment w1 w2 Data Height  1H-H 6.704 6.322 9224233  2H-H 6.678 6.372 9898637  3H-H 6.419 4.456 20015776  4H-H 6.368 6.681 11333652  5H-H 6.320 6.706 11307411  6H-H 6.171 5.044 134583264  7H-H 6.043 4.952 12934876  8H-H 5.980 2.030 80531096  9H-H 5.980 2.021 105609248  10H-H 5.881 1.963 81350000  11H-H 5.881 1.952 75881512  12H-H 5.701 5.700 215204304  13H-H 5.700 5.590 18340750  14H-H 5.699 5.575 15251625  15H-H 5.582 5.701 24882492  16H-H 5.583 4.930 32200604  17H-H 5.583 4.269 54166860  18H-H 5.582 4.260 100437000  19H-H 5.582 4.251 50904084  20H-H 5.335 4.478 54908116  21H-H 5.335 4.462 48000284  22H-H 5.334 4.100 20768318  23H-H 5.048 6.174 89161792  24H-H 4.949 4.792 11766272  25H-H 4.950 4.782 9033592  26H-H 4.930 5.583 17147768  27H-H 4.929 4.269 30517720  28H-H 4.927 4.251 28975240  29H-H 4.901 4.390 27806448  30H-H 4.900 4.373 34077108  31H-H 4.831 4.790 99849200  32H-H 4.820 4.310 7275091  33H-H 4.819 4.296 7797132  34H-H 4.822 4.157 13857122  35H-H 4.821 4.138 18211468  36H-H 4.794 4.953 30630578  37H-H 4.787 4.827 86722272  38H-H 4.568 4.109 17535982  39H-H 4.567 4.093 15653847  40H-H 4.542 4.466 32239438  41H-H 4.522 4.453 145233152  42H-H 4.517 4.205 7769614  43H-H 4.523 3.952 73572400  44H-H 4.507 4.436 11835048  45H-H 4.468 5.337 79724560  46H-H 4.468 4.099 83781344  47H-H 4.461 3.954 44029948  48H-H 4.454 6.528 9453154  49H-H 4.454 6.418 79266688  50H-H 4.450 4.381 13566321  51H-H 4.449 4.321 8581714  52H-H 4.444 4.214 29697092  53H-H 4.445 4.205 23505830  54H-H 4.443 4.195 23169768  55H-H 4.382 4.902 75968808  56H-H 4.380 4.214 34194940  57H-H 4.379 4.204 34462264  58H-H 4.380 4.195 34413552  59H-H 4.302 4.823 16076232  60H-H 4.302 4.148 221634448  61H-H 4.262 5.582 80620088  62H-H 4.261 4.930 59383108  63H-H 4.203 4.900 12911051  64H-H 4.204 4.433 20195584  65H-H 4.150 4.832 17371252  66H-H 4.153 4.819 18323128  67H-H 4.149 4.305 216562864  68H-H 4.099 5.337 25030224  69H-H 4.100 4.571 36121208  70H-H 4.101 4.472 68659520  71H-H 4.095 4.460 36864516  72H-H 4.028 4.468 7964866  73H-H 4.028 4.458 8388422  74H-H 3.961 4.463 41897776  75H-H 3.953 4.539 34935532  76H-H 3.952 4.523 50083884  77H-H 3.953 4.447 24637258  78H-H 3.893 1.162 59572844  79H-H 3.887 1.175 95668312  80H-H 3.766 3.516 11119055  81H-H 3.744 3.494 6884830  82H-H 3.598 3.413 146852352  83H-H 3.598 3.404 128194976  84H-H 3.517 3.767 10865892  85H-H 3.508 3.759 9535602  86H-H 3.480 3.741 8054603  87H-H 3.475 3.734 6268863  88H-H 3.409 3.599 107289744  89H-H 3.406 3.592 110786000  90H-H 3.229 2.109 12003553  91H-H 2.110 1.838 8593652  92H-H 2.107 1.822 11474128  93H-H 2.107 1.811 10279236  94H-H 2.105 1.794 8116300  95H-H 2.029 5.992 47626580  96H-H 2.027 5.984 47768532  97H-H 2.026 5.972 51874608  98H-H 2.030 2.118 7536440  99H-H 2.027 1.919 89540200 100H-H 2.024 5.964 41580316 101H-H 1.959 5.891 49355596 102H-H 1.955 5.882 46234528 103H-H 1.956 5.870 51174608 104H-H 1.954 1.848 77092128 105H-H 1.923 2.030 54674196 106H-H 1.917 2.020 54160620 107H-H 1.848 1.958 62600468 108H-H 1.834 1.756 16637570 109H-H 1.829 1.732 26336268 110H-H 1.828 1.711 15651960 111H-H 1.835 1.677 25025304 112H-H 1.830 1.553 106561752 113H-H 1.828 1.536 92597192 114H-H 1.820 3.231 15325426 115H-H 1.819 3.220 13169861 116H-H 1.818 2.114 14925469 117H-H 1.818 2.106 16020545 118H-H 1.742 1.848 24286072 119H-H 1.745 1.827 35995120 120H-H 1.669 1.827 14851102 121H-H 1.546 1.834 96039856 122H-H 1.543 1.819 72780528 123H-H 1.545 1.270 7218780 124H-H 1.546 1.255 36723868 125H-H 1.544 1.241 22604678 126H-H 1.476 1.271 6846019 127H-H 1.477 1.255 10952480 128H-H 1.471 1.242 8182750 129H-H 1.474 0.727 7531610 130H-H 1.348 0.812 23199942 131H-H 1.348 0.796 27081584 132H-H 1.344 0.784 22663138 133H-H 1.260 1.540 12084268 134H-H 1.263 1.489 14416738 135H-H 1.263 1.474 17048864 136H-H 1.267 0.736 16594936 137H-H 1.266 0.719 16949332 138H-H 1.252 1.558 13171360 139H-H 1.250 1.525 19080828 140H-H 1.237 0.837 11506187 141H-H 1.167 3.901 86146744 142H-H 1.167 3.877 80709560 143H-H 0.840 1.234 8462904 144H-H 0.798 1.351 39178912 145H-H 0.798 1.342 32789168 146H-H 0.730 1.482 11378742 147H-H 0.731 1.270 11791943 148H-H 0.729 1.255 15935769 149H-H 4.460 4.531 92968608 150H-H 4.203 4.382 45014720 151H-H 4.203 4.373 32816148 152H-H 1.795 1.675 4628933

TABLE 6.5. Proton NMR chemical shift data of Y1 FREQUENCY # ADDRESS [Hz] [PPM] INTENSITY 1 5803.6 5312.007 8.8510 1.05 2 6201.8 5224.701 8.7055 180.00 3 6517.2 5155.558 8.5903 0.09 4 6612.7 5134.626 8.5554 1.05 5 8992.9 4612.799 7.6859 0.41 6 9356.8 4533.029 7.5530 62.60 7 9733.1 4450.514 7.4155 0.40 8 9952.7 4402.383 7.3353 0.13 9 9996.2 4392.832 7.3194 0.90 10 10361.5 4312.746 7.1860 134.51 11 10641.7 4251.327 7.0837 0.05 12 10739.1 4229.980 7.0481 0.87 13 12483.6 3847.521 6.4108 0.10 14 12504.0 3843.034 6.4033 0.10 15 13117.1 3708.630 6.1794 0.31 16 13161.7 3698.843 6.1631 0.31 17 13503.5 3623.905 6.0382 0.38 18 13617.2 3598.988 5.9967 0.08 19 13649.9 3591.811 5.9848 0.19 20 13679.2 3585.404 5.9741 0.18 21 13712.9 3577.998 5.9617 0.08 22 13886.8 3539.884 5.8982 0.09 23 13920.0 3532.594 5.8861 0.19 24 13952.5 3525.479 5.8742 0.17 25 13983.6 3518.658 5.8629 0.06 26 14413.7 3424.364 5.7058 0.36 27 14428.7 3421.080 5.7003 0.36 28 14720.0 3357.206 5.5939 0.19 29 14735.6 3353.801 5.5882 0.18 30 14766.4 3347.042 5.5769 0.20 31 14781.9 3343.652 5.5713 0.17 32 15028.7 3289.533 5.4811 0.05 33 15343.4 3220.543 5.3661 0.33 34 15404.6 3207.131 5.3438 0.25 35 15439.6 3199.461 5.3310 0.25 36 16193.8 3034.111 5.0555 0.14 37 16238.8 3024.240 5.0391 0.15 38 16381.2 2993.014 4.9870 0.17 39 16518.3 2962.967 4.9370 0.12 40 16553.7 2955.209 4.9240 0.12 41 16599.3 2945.198 4.9074 0.08 42 16633.5 2937.700 4.8949 0.08 43 16843.2 2891.736 4.8183 0.18 44 16905.3 2878.117 4.7956 0.17 45 16927.0 2873.370 4.7877 0.19 46 17523.6 2742.566 4.5697 0.33 47 17586.3 2728.818 4.5468 0.18 48 17617.6 2721.965 4.5354 0.23 49 17635.4 2718.066 4.5289 0.31 50 17665.9 2711.371 4.5177 0.32 51 17716.2 2700.349 4.4994 0.21 52 17760.5 2690.632 4.4832 0.20 53 17810.9 2679.588 4.4648 0.55 54 17835.7 2674.141 4.4557 0.72 55 17857.9 2669.267 4.4476 0.54 56 18007.8 2636.409 4.3928 0.17 57 18046.2 2627.986 4.3788 0.26 58 18083.2 2619.886 4.3653 0.19 59 18189.9 2596.493 4.3263 0.11 60 18233.6 2586.912 4.3104 0.27 61 18277.8 2577.209 4.2942 0.31 62 18331.5 2565.443 4.2746 0.21 63 18367.6 2557.542 4.2614 0.23 64 18412.4 2547.721 4.2451 0.19 65 18458.8 2537.540 4.2281 0.10 66 18491.9 2530.283 4.2160 0.21 67 18532.6 2521.359 4.2011 0.28 68 18567.2 2513.770 4.1885 0.14 69 18645.4 2496.625 4.1599 0.25 70 18666.5 2491.997 4.1522 0.25 71 18699.9 2484.686 4.1400 0.24 72 18721.5 2479.943 4.1321 0.19 73 18778.7 2467.413 4.1113 0.20 74 18792.8 2464.305 4.1061 0.20 75 18821.9 2457.939 4.0955 0.19 76 18835.7 2454.909 4.0904 0.18 77 19186.2 2378.063 3.9624 0.17 78 19213.7 2372.029 3.9523 0.29 79 19239.6 2366.350 3.9429 0.16 80 19338.7 2344.635 3.9067 0.08 81 19366.7 2338.490 3.8964 0.22 82 19396.9 2331.872 3.8854 0.21 83 19428.2 2325.007 3.8740 0.06 84 20015.2 2196.324 3.6596 0.05 85 20048.6 2188.994 3.6474 0.08 86 20131.1 2170.900 3.6172 0.13 87 20161.7 2164.211 3.6061 0.28 88 20208.1 2154.040 3.5891 0.31 89 20679.1 2050.765 3.4170 0.24 90 20727.6 2040.135 3.3993 0.23 91 20782.4 2028.119 3.3793 0.08 92 21179.2 1941.123 3.2343 0.14 93 21213.6 1933.579 3.2218 0.13 94 21586.4 1851.862 3.0856 0.07 95 21649.4 1838.049 3.0626 0.09 96 21715.8 1823.490 3.0383 0.67 97 21747.5 1816.527 3.0267 0.23 98 23153.9 1508.199 2.5130 0.06 99 23693.5 1389.916 2.3159 2.02 100 24231.4 1271.971 2.1194 0.22 101 24282.2 1260.831 2.1008 0.22 102 24341.3 1247.878 2.0792 0.16 103 24413.6 1232.024 2.0528 0.15 104 24464.0 1220.989 2.0344 0.83 105 24468.7 1219.963 2.0327 0.83 106 24496.7 1213.827 2.0225 0.82 107 24540.5 1204.212 2.0065 0.24 108 24614.2 1188.066 1.9796 0.24 109 24658.3 1178.391 1.9635 0.88 110 24686.0 1172.311 1.9533 0.86 111 24690.9 1171.249 1.9516 0.85 112 24774.5 1152.908 1.9210 1.65 113 24824.7 1141.905 1.9027 0.22 114 24966.5 1110.815 1.8509 2.07 115 25021.9 1098.679 1.8306 2.09 116 25154.1 1069.689 1.7823 0.22 117 25213.5 1056.676 1.7607 0.20 118 25280.4 1042.010 1.7362 0.33 119 25347.2 1027.363 1.7118 0.24 120 25396.2 1016.604 1.6939 0.38 121 25591.3 973.839 1.6226 0.16 122 25626.4 966.149 1.6098 0.22 123 25673.7 955.785 1.5926 0.16 124 25759.1 937.061 1.5614 0.45 125 25837.0 919.968 1.5329 0.32 126 25932.6 899.023 1.4980 0.22 127 25995.9 885.130 1.4748 0.28 128 26142.6 852.967 1.4212 1.52 129 26284.2 821.936 1.3695 1.99 130 26355.3 806.354 1.3436 0.49 131 26405.2 795.414 1.3253 0.53 132 26437.0 788.423 1.3137 0.61 133 26495.4 775.620 1.2924 1.83 134 26547.6 764.186 1.2733 0.62 135 26578.6 757.400 1.2620 0.75 136 26635.0 745.018 1.2414 0.66 137 26710.2 728.529 1.2139 0.35 138 26742.2 721.515 1.2022 0.34 139 26774.1 714.532 1.1906 0.38 140 26818.0 704.909 1.1745 1.47 141 26852.6 697.329 1.1619 2.39 142 26939.0 678.382 1.1303 0.28 143 26967.1 672.226 1.1201 0.28 144 26987.0 667.851 1.1128 0.31 145 27035.8 657.144 1.0949 0.25 146 27059.9 651.863 1.0861 0.21 147 27091.3 644.992 1.0747 0.18 148 27116.3 639.514 1.0656 0.13 149 27306.1 597.891 0.9962 0.21 150 27331.8 592.271 0.9869 0.20 151 27362.1 585.622 0.9758 0.18 152 27426.7 571.457 0.9522 0.07 153 27465.2 563.025 0.9381 0.12 154 27550.1 544.395 0.9071 0.06 155 27604.4 532.507 0.8873 0.09 156 27723.7 506.347 0.8437 0.23 157 27778.4 494.349 0.8237 0.48 158 27825.7 483.975 0.8064 1.88 159 27892.5 469.335 0.7820 1.79 160 27999.1 445.968 0.7431 0.28 161 28054.1 433.917 0.7230 0.23 162 28134.1 416.375 0.6938 0.08 163 28228.5 395.678 0.6593 0.07 164 28262.5 388.209 0.6468 0.09

TABLE 8.2. 2D NMR (HMQC) of R1 peaklist. ADDRESS FREQUENCY row [Hz]F1 [PPM]F1 # col [Hz]F2 [PPM]F2 INTENSITY 1 151 20624.836 136.6695 4.88 397 3494.332 5.8223 2 157 20461.906 135.5898 67.68 220 4534.469 7.5554 3 234 18646.779 123.5620 100.00 258 4313.193 7.1867 4 234 18646.779 123.5620 9.18 445 3213.863 5.3550 5 343 16069.902 106.4864 7.58 496 2913.367 4.8543 6 343 16069.902 106.4864 7.61 498 2904.202 4.8390 7 350 15895.188 105.3287 7.99 471 3059.891 5.0985 8 350 15895.188 105.3287 9.11 473 3050.642 5.0830 9 350 15895.188 105.3287 8.79 481 3004.427 5.0060 10 363 15605.384 103.4083 7.96 510 2835.541 4.7246 11 380 15192.972 100.6755 16.30 326 3912.206 6.5186 12 454 13447.552 89.1095 4.35 656 1974.610 3.2901 13 454 13447.552 89.1095 4.45 659 1958.017 3.2625 14 515 12025.116 79.6838 4.85 561 2533.049 4.2206 15 518 11934.442 79.0830 4.52 389 3540.938 5.9000 16 524 11802.268 78.2071 12.80 562 2525.183 4.2075 17 524 11802.268 78.2071 11.92 594 2340.880 3.9004 18 534 11563.501 76.6250 4.43 571 2474.88 34.1237 19 534 11563.501 76.6250 3.52 588 2372.543 3.9532 20 534 11563.501 76.6250 5.94 610 2246.165 3.7426 21 534 11563.501 76.6250 6.60 612 2234.362 3.7229 22 540 11437.312 75.7888 20.96 574 2457.347 4.0945 23 545 11309.472 74.9417 5.29 553 2580.628 4.2999 24 545 11309.472 74.9417 6.15 555 2569.264 4.2810 25 545 11309.472 74.9417 3.23 561 2533.966 4.2222 26 545 11309.472 74.9417 9.38 582 2411.457 4.0180 27 551 11175.793 74.0558 3.74 550 2597.570 4.3281 28 551 11175.793 74.0558 7.04 552 2585.925 4.3087 29 551 11175.793 74.0558 3.84 554 2574.515 4.2897 30 556 11047.378 73.2049 5.90 586 2386.166 3.9759 31 562 10907.159 72.2758 15.78 511 2826.203 4.7091 32 562 10907.159 72.2758 5.35 520 2777.020 4.6271 33 567 10784.600 71.4636 12.61 562 2525.038 4.2073 34 567 10784.600 71.4636 4.33 567 2497.664 4.1617 35 577 10543.526 69.8662 4.00 497 2908.530 4.8463 36 577 10543.526 69.8662 3.60 500 2892.560 4.8196 37 577 10543.526 69.8662 5.98 514 2810.206 4.6824 38 577 10543.526 69.8662 5.96 516 2796.413 4.6594 39 577 10543.526 69.8662 6.72 548 2609.404 4.3478 40 624 9434.661 62.5183 11.62 532 2705.029 4.5072 41 624 9434.661 62.5183 8.01 534 2689.727 4.4817 42 624 9434.661 62.5183 7.54 547 2616.577 4.3598 43 669 8381.180 55.5375 4.80 919 431.689 0.7193 44 669 8381.180 55.5375 5.21 922 414.881 0.6913 45 719 7203.831 47.7358 3.32 827 972.057 1.6197 46 728 6986.542 46.2960 3.85 777 1265.417 2.1085 47 728 6986.542 46.2960 3.19 859 784.626 1.3074 48 728 6986.542 46.2960 3.45 862 764.970 1.2746 49 759 6273.788 41.5729 3.64 724 1575.188 2.6246 50 759 6273.788 41.5729 728 1556.033 2.5927 3.54 51 817 4904.791 32.5014 9.21 849 844.868 1.4077 52 831 4556.851 30.1957 40.19 868 732.276 1.2201 53 846 4212.870 27.9164 45.73 870 719.170 1.1983 54 858 3932.408 26.0579 45.69 865 749.675 1.2491 55 893 3102.798 20.5605 37.60 808 1085.904 1.8094 56 900 2935.044 19.4489 42.87 864 755.857 1.2594 57 905 2821.322 18.6953 25.73 808 1086.381 1.8102 58 918 2523.121 16.7193 36.59 883 644.770 1.0743 59 918 2523.121 16.7193 43.39 895 571.319 0.9519 60 924 2368.333 15.6936 21.68 794 1163.335 1.9384 61 924 2368.333 15.6936 45.90 895 573.050 0.9548

TABLE 8.3. HMBC R1 peaklist DU = C: /Bruker/XWIN-NMR, USER = guest, NAME = chan, EXPNO = F1PLO = 173.650 ppm, F1PHI = 11.502 ppm, F2PLO = 6.832 ppm, F2IMI = 5.00 cm, MAXI = 10000.00 cm, PC = 3.000 ADDRESS FREQUENCY row [Hz]F1 [PPM]F1 # col [Hz]F2 [PPM]F2 INTENSITY 1 166 25330.719 167.8561 11.00 389 3542.372 5.9024 2 166 25330.719 167.8561 37.94 808 1085.572 1.8088 3 293 21569.176 142.9299 39.69 865 750.004 1.2497 4 325 20624.461 136.6697 54.15 795 1160.826 1.9342 5 325 20624.461 136.6697 67.95 808 1085.191 1.8082 6 366 19424.748 128.7197 54.29 795 1161.341 1.9351 7 366 19424.748 128.7197 100.00 808 1085.293 1.8083 8 479 16074.168 106.5168 17.03 587 2382.396 3.9696 9 485 15911.199 105.4369 13.64 582 2411.570 4.0182 10 495 15610.658 103.4453 7.40 559 2544.112 4.2391 11 495 15610.658 103.4453 7.72 574 2457.717 4.0951 12 509 15196.733 100.7024 10.46 311 3999.566 6.6642 13 509 15196.733 100.7024 10.97 341 3825.973 6.3749 14 568 13447.739 89.1126 13.78 497 2908.018 4.8454 15 568 13447.739 89.1126 52.71 870 718.459 1.1971 16 568 13447.739 89.1126 33.62 896 570.271 0.9502 17 616 12037.521 79.7676 10.39 560 2540.187 4.2325 18 616 12037.521 79.7676 12.77 567 2496.423 4.1596 19 620 11934.375 79.0841 16.69 557 2555.670 4.2583 20 620 11934.375 79.0841 18.17 586 2384.236 3.9727 21 624 11794.329 78.1561 29.22 561 2532.465 4.2196 22 624 11794.329 78.1561 13.90 581 2415.675 4.0251 23 632 11561.078 76.6104 11.27 554 2575.363 4.2911 24 632 11561.078 76.6104 8.95 861 774.214 1.2900 25 632 11561.078 76.6104 31.05 864 757.631 1.2624 26 632 11561.078 76.6104 52.22 868 733.017 1.2214 27 640 11319.147 75.0073 14.09 562 2529.498 4.2147 28 640 11319.147 75.0073 12.03 574 2457.979 4.0955 29 640 11319.147 75.0073 7.35 611 2238.440 3.7297 30 645 11173.489 74.0421 5.08 511 2826.331 4.7093 31 645 11173.489 74.0421 27.89 807 1087.742 1.8124 32 650 11046.945 73.2035 14.57 389 3541.020 5.9001 33 655 10904.058 72.2566 23.27 326 3913.536 6.5208 34 655 10904.058 72.2566 9.96 552 2586.768 4.3101 35 655 10904.058 72.2566 6.06 563 2521.903 4.2021 36 659 10780.204 71.4359 6.93 532 2702.900 4.5036 37 659 10780.204 71.4359 8.19 534 2692.127 4.4857 38 659 10780.204 71.4359 48.21 563 2520.533 4.1998 39 667 10532.287 69.7931 11.46 472 3054.495 5.0895 40 667 10532.287 69.7931 18.84 480 3007.639 5.0114 41 671 10425.301 69.0841 32.61 326 3913.495 6.5208 42 671 10425.301 69.0841 7.93 552 2586.054 4.3089 43 671 10425.301 69.0841 58.15 807 1088.313 1.8134 44 705 9430.492 62.4919 5.37 560 2539.835 4.2319 45 740 8372.175 55.4789 8.08 870 718.660 1.19743 46 740 8372.175 55.4789 44.50 895 571.407 0.9521 47 780 7199.307 47.7068 27.47 883 644.628 1.0741 48 780 7199.307 47.7068 31.41 895 572.447 0.9538 49 788 6981.523 46.2636 51.63 864 757.767 1.2626 50 788 6981.523 46.2636 78.63 868 732.960 1.2213 51 804 6495.941 43.0459 8.82 574 2458.027 4.0956 52 804 6495.941 43.0459 8.89 861 774.879 1.2911 53 804 6495.941 43.0459 6.55 867 737.966 1.2296 54 810 6313.906 41.8396 8.33 445 3213.454 5.3543 55 810 6313.906 41.8396 5.40 755 1397.464 2.3285 56 810 6313.906 41.8396 42.24 865 749.894 1.2495 57 810 6313.906 41.8396 52.86 883 644.620 1.0741 58 814 6189.256 41.0136 6.31 865 749.287 1.2485 59 814 6189.256 41.0136 8.62 883 643.362 1.0720 60 817 6120.849 40.5603 15.69 865 749.159 1.2483 61 817 6120.849 40.5603 17.94 883 644.286 1.0735 62 817 6120.849 40.5603 6.27 896 570.189 0.9501 63 819 6053.888 40.1166 43.77 865 749.605 1.2490 64 819 6053.888 40.1166 47.98 883 644.397 1.0737 65 819 6053.888 40.1166 8.91 896 570.067 0.9499 66 824 5921.430 39.2388 5.80 864 754.133 1.2566 67 824 5921.430 39.2388 70.97 870 718.264 1.1968 68 824 5921.430 39.2388 65.30 896 569.449 0.9488 69 824 5921.430 39.2388 6.70 920 427.451 0.7122 70 824 5921.430 39.2388 7.41 922 414.558 0.6907 71 827 5818.316 38.5556 8.71 870 718.161 1.1966 72 827 5818.316 38.5556 35.78 895 572.960 0.9547 73 829 5749.308 38.0983 8.61 896 570.060 0.9498 74 837 5529.916 36.6444 5.22 829 959.437 1.5986 75 837 5529.916 36.6444 12.15 864 755.929 1.2595 76 837 5529.916 36.6444 8.77 868 732.618 1.2207 77 837 5529.916 36.6444 51.81 895 572.369 0.9537 78 837 5529.916 36.6444 5.36 920 427.331 0.7120 79 837 5529.916 36.6444 6.76 922 414.571 0.6908 80 840 5443.605 36.0725 5.25 777 1264.89 12.1076 81 840 5443.605 36.0725 68.88 864 757.333 1.2619 82 840 5443.605 36.0725 83.66 868 732.776 1.2210 83 840 5443.605 36.0725 11.13 895 571.988 0.9531 84 858 4903.620 32.4942 38.16 883 644.423 1.0738 85 870 4556.862 30.1964 8.07 611 2240.976 3.7340 86 870 4556.862 30.1964 18.78 857 794.987 1.3246 87 870 4556.862 30.1964 79.89 864 756.793 1.2610 88 870 4556.862 30.1964 18.43 878 670.310 1.1169 89 872 4487.079 29.7340 16.38 864 756.125 1.2599 90 874 4422.445 29.3057 8.16 863 758.384 1.2636 91 877 4356.193 28.8667 8.79 864 755.084 1.2581 92 877 4356.193 28.8667 6.22 896 569.898 0.9496 93 882 4211.267 27.9063 19.55 860 779.212 1.2983 94 882 4211.267 27.9063 5.59 864 754.574 1.2573 95 882 4211.267 27.9063 19.01 881 654.994 1.0914 96 882 4211.267 27.9063 87.84 896 570.162 0.9500 97 891 3938.165 26.0966 19.50 854 812.119 1.3532 98 891 3938.165 26.0966 10.44 865 751.828 1.2527 99 891 3938.165 26.0966 17.89 876 686.003 1.1430 100 891 3938.165 26.0966 5.20 896 569.852 0.9495 101 893 3876.630 25.6888 40.70 865 749.874 1.2495 102 893 3876.630 25.6888 5.01 876 686.400 1.1437 103 919 3095.315 20.5114 7.58 398 3488.142 5.8120 104 919 3095.315 20.5114 9.43 797 1150.511 1.9170 105 919 3095.315 20.5114 9.18 818 1022.904 1.7044 106 919 3095.315 20.5114 5.53 868 732.855 1.2211 107 925 2937.158 19.4633 13.27 611 2240.910 3.7339 108 925 2937.158 19.4633 11.35 777 1265.381 2.1084 109 925 2937.158 19.4633 5.80 780 1248.250 2.0799 110 925 2937.158 19.4633 23.23 853 818.856 1.3644 111 925 2937.158 19.4633 89.32 868 733.355 1.2219 112 925 2937.158 19.4633 20.36 875 693.473 1.1555 113 932 2710.756 17.9630 6.54 554 2574.880 4.2903 114 932 2710.756 17.9630 5.24 849 843.916 1.4062 115 932 2710.756 17.9630 6.51 868 732.674 1.2208 116 939 2527.765 16.7504 6.70 829 960.874 1.6010 117 939 2527.765 16.7504 83.68 870 718.686 1.1975 118 939 2527.765 16.7504 21.97 885 631.287 1.0519 119 939 2527.765 16.7504 14.30 893 583.038 0.9715 120 939 2527.765 16.7504 22.28 906 506.673 0.8442 121 939 2527.765 16.7504 7.71 920 426.044 0.7099 122 939 2527.765 16.7504 10.74 922 414.883 0.6913 123 944 2376.340 15.7470 9.28 784 1224.570 2.0404 124 944 2376.340 15.7470 8.54 806 1096.659 1.8273 125 944 2376.340 15.7470 6.60 870 718.378 1.1970 126 944 2376.340 15.7470 19.99 885 633.381 1.0554 127 944 2376.340 15.7470 20.05 906 509.452 0.8489 128 944 2376.340 15.7470 8.96 920 426.988 0.7115 129 944 2376.340 15.7470 10.34 922 415.107 0.6917

TABLE 8.4. 2D NMR (cosy) of R1 peak list Assignment w1 w2 Data Height 1 6.521 6.522 257252096 2 6.522 4.711 7840252 3 6.129 0.959 3905950 4 5.898 5.901 32367934 5 5.902 4.847 10836326 6 5.902 4.258 10138593 7 5.899 4.127 9360363 8 5.906 3.975 16282743 9 5.821 1.810 7215536 10 5.817 5.822 31028110 11 5.817 1.944 43526536 12 5.816 1.929 44815556 13 5.355 5.355 178856592 14 5.094 5.102 109124320 15 5.094 5.085 116649128 16 5.092 4.205 15712139 17 5.093 4.048 38195456 18 5.011 5.021 112612352 19 5.010 5.004 121053544 20 5.008 4.210 14216868 21 5.011 4.021 38155584 22 4.847 5.913 5875469 23 4.845 5.900 4787344 24 4.845 5.889 7478266 25 4.841 4.855 118994368 26 4.836 4.823 47239140 27 4.836 4.369 92117104 28 4.831 4.134 9652155 29 4.834 4.116 7044446 30 4.844 3.987 44245188 31 4.845 3.962 44209660 32 4.798 4.795 12493983 33 4.795 4.621 15346245 34 4.796 1.821 57265952 35 4.796 1.809 75309312 36 4.792 4.312 21750568 37 4.753 4.360 24472454 38 4.725 4.733 72665064 39 4.721 4.719 99191696 40 4.727 4.226 50380324 41 4.709 6.523 22903380 42 4.702 4.629 20594276 43 4.707 4.612 19038358 44 4.706 4.326 11671381 45 4.670 4.688 34985344 46 4.669 4.660 40038520 47 4.669 4.355 94428136 48 4.670 3.958 8587019 49 4.672 3.941 7812038 50 4.620 4.800 8615562 51 4.619 4.711 21326744 52 4.618 4.633 29866052 53 4.619 4.607 44064012 54 4.621 4.312 23669784 55 4.584 4.355 27276566 56 4.566 4.365 23402122 57 4.492 4.513 55339136 58 4.491 4.482 71439344 59 4.496 4.370 164934224 60 4.495 4.360 174062528 61 4.499 3.912 17609992 62 4.498 3.896 17924064 63 4.377 4.349 24720128 64 4.365 4.843 97382152 65 4.364 4.829 127752656 66 4.356 4.680 67867680 67 4.355 4.667 81378376 68 4.361 4.491 211941920 69 4.374 4.393 38442384 70 4.364 4.372 27706436 71 4.358 4.335 31327086 72 4.364 4.137 14876345 73 4.363 4.113 13081764 74 4.355 3.960 10728486 75 4.357 3.940 9552408 76 4.371 3.913 20295026 77 4.372 3.895 17021364 78 4.309 4.799 25905518 79 4.309 4.619 43016120 80 4.306 4.310 56206892 81 4.294 3.742 80581928 82 4.295 3.731 101483432 83 4.280 4.354 9482896 84 4.251 5.915 17374606 85 4.250 5.901 10701927 86 4.251 5.887 17140758 87 4.253 4.259 32444480 88 4.258 4.125 31047850 89 4.257 3.976 5638105 90 4.229 4.730 34642484 91 4.207 5.092 13039638 92 4.209 5.012 11226875 93 4.217 4.179 33544654 94 4.209 4.058 28007252 95 4.207 4.047 46281960 96 4.211 4.020 33876260 97 4.207 4.005 21146824 98 4.210 3.904 80923600 99 4.204 4.365 9066176 100 4.202 4.032 40055832 101 4.167 3.951 18178542 102 4.150 4.209 11484229 103 4.126 5.912 6719130 104 4.122 5.890 7671593 105 4.126 4.378 9045784 106 4.127 4.273 34269312 107 4.119 4.258 32734018 108 4.133 4.246 34046856 109 4.131 4.140 18941800 110 4.116 4.355 11770206 111 4.092 4.096 279326848 112 4.043 5.095 84940104 113 4.035 4.208 50268828 114 4.042 4.183 15655260 115 4.043 4.064 26875002 116 4.019 5.015 77663464 117 4.015 4.241 9036684 118 4.027 4.226 11811450 119 4.016 4.037 19835598 120 3.973 5.915 24059896 121 3.974 5.901 20609464 122 3.973 5.887 22835978 123 3.975 4.853 37923332 124 3.977 3.995 18827896 125 3.949 4.682 5809583 126 3.950 4.371 10668304 127 3.953 4.348 15916606 128 3.949 4.175 18189762 129 3.948 4.161 30027716 130 3.953 4.149 19945408 131 3.940 3.966 14571032 132 3.903 4.505 10048077 133 3.903 4.488 14760281 134 3.903 4.387 19898852 135 3.912 4.342 16459215 136 3.904 4.239 12064081 137 3.907 4.214 108106008 138 3.898 4.364 32756190 139 3.900 4.199 27604272 140 3.901 3.917 43085776 141 3.897 3.888 38415896 142 3.735 4.300 93317560 143 3.736 4.290 98467152 144 3.736 3.748 65327612 145 3.735 3.725 50388764 146 3.270 3.283 17369858 147 3.269 3.267 22139430 148 3.271 2.354 5853844 149 3.271 2.342 7970904 150 3.271 1.912 5107326 151 3.271 1.896 5093256 152 2.602 2.631 5486520 153 2.600 2.598 5094291 154 2.601 2.590 7143968 155 2.602 2.132 14357642 156 2.602 2.109 26185532 157 2.601 2.086 14739034 158 2.603 1.289 14662965 159 2.341 3.287 5980422 160 2.342 3.264 5054491 161 2.334 2.360 5303651 162 2.336 1.932 6500135 163 2.334 1.872 7760766 164 2.325 2.341 13008529 165 2.325 2.315 11101831 166 2.328 1.923 6345431 167 2.326 1.895 9356538 168 2.289 2.294 14262412 169 2.186 2.193 4223454 170 2.103 2.620 30164346 171 2.103 2.598 27444406 172 2.101 2.109 7249900 173 2.105 1.307 22762886 174 2.102 1.300 18897670 175 2.103 1.279 18250726 176 2.070 2.078 4104108 177 1.967 1.969 13308390 178 1.977 1.609 7894784 179 1.956 1.909 11551598 180 1.961 1.874 10608534 181 1.965 1.625 5946444 182 1.931 5.834 39974876 183 1.931 5.824 42157648 184 1.932 5.812 40474008 185 1.931 5.798 30554264 186 1.931 2.320 12384753 187 1.931 1.942 228192416 188 1.931 1.810 59524928 189 1.913 2.339 13306613 190 1.902 2.001 6772636 191 1.895 3.287 9744711 192 1.896 3.264 9357915 193 1.897 2.362 8772959 194 1.886 1.973 16266749 195 1.885 1.885 5269322 196 1.882 1.616 15011650 197 1.883 1.242 8077304 198 1.878 1.219 8767326 199 1.818 5.840 3029832 200 1.805 5.829 3553817 201 1.804 5.808 3418502 202 1.805 4.822 22320414 203 1.805 4.813 19315348 204 1.806 4.797 27156710 205 1.806 4.785 23402710 206 1.806 4.774 22620674 207 1.806 4.322 15274810 208 1.806 4.312 11189881 209 1.806 4.302 17586586 210 1.802 1.943 42426312 211 1.804 1.932 30620986 212 1.804 1.810 1349507072 213 1.776 1.778 8674281 214 1.703 1.710 5954890 215 1.696 1.702 9409535 216 1.613 1.911 8734371 217 1.611 1.635 17314310 218 1.609 1.610 29144662 219 1.609 1.599 16356248 220 1.613 1.032 9463035 221 1.614 1.017 7358422 222 1.613 0.991 9572058 223 1.406 1.303 9782705 224 1.395 1.406 70684776 225 1.393 1.385 22170084 226 1.390 1.293 11202919 227 1.307 1.318 7420988 228 1.285 2.609 28951250 229 1.284 2.133 16431712 230 1.285 2.110 23106420 231 1.283 2.083 14481833 232 1.279 1.407 11364364 233 1.288 1.384 6974150 234 1.286 1.299 18845462 235 1.288 0.712 21876642 236 1.288 0.696 27076318 237 1.251 1.260 768046912 238 1.221 1.927 5903658 239 1.217 1.223 1002405376 240 1.188 1.197 664805376 241 1.168 1.171 16019256 242 1.137 1.145 30540644 243 1.113 1.120 37708240 244 1.070 1.077 775147520 245 1.058 1.058 17850510 246 1.004 2.349 5175155 247 1.003 1.916 6850918 248 1.003 1.892 5963474 249 1.005 1.630 21215516 250 1.005 1.610 23547650 251 1.000 1.008 25525576 252 1.016 0.959 33680632 253 0.985 0.999 11598959 254 0.963 0.985 13491636 255 0.944 0.953 1674528384 256 0.912 0.912 21859108 257 0.872 0.878 12589871 258 0.697 0.716 28731108 259 0.699 0.693 23421302 260 0.694 1.297 7870457 261 0.692 1.289 8126754

TABLE 8.5 R1-proton-NMR-peaklist FREQUENCY # ADDRESS [Hz] [PPM] INTENSITY 1 13957.6 3913.934 6.5215 20.81 2 15614.0 3550.810 5.9164 7.03 3 15656.8 3541.408 5.9008 14.16 4 15699.7 3532.016 5.8851 7.43 5 15839.6 3501.350 5.8340 2.56 6 15872.2 3494.204 5.8221 6.47 7 15905.1 3486.992 5.8101 6.47 8 15932.8 3480.916 5.8000 2.49 9 17152.3 3213.554 5.3545 12.90 10 17853.4 3059.848 5.0984 17.52 11 17888.8 3052.093 5.0855 18.03 12 18074.9 3011.286 5.0175 16.80 13 18110.2 3003.544 5.0046 17.26 14 18524.3 2912.751 4.8533 15.71 15 18558.4 2905.282 4.8408 20.27 16 18597.3 2896.749 4.8266 12.17 17 18638.6 2887.709 4.8116 5.88 18 18667.2 2881.421 4.8011 6.29 19 18680.9 2878.427 4.7961 6.65 20 18709.4 2872.187 4.7857 5.58 21 18857.7 2839.672 4.7315 13.15 22 18891.0 2832.356 4.7193 15.61 23 18902.5 2829.844 4.7151 13.51 24 18918.1 2826.431 4.7095 13.25 25 18996.6 2809.208 4.6808 8.60 26 19038.5 2800.032 4.6655 9.32 27 19136.8 2778.471 4.6296 7.89 28 19151.7 2775.208 4.6241 6.99 29 19179.0 2769.220 4.6141 8.39 30 19194.1 2765.907 4.6086 7.85 31 19474.8 2704.369 4.5061 16.52 32 19515.8 2695.385 4.4911 14.33 33 19529.0 2692.503 4.4863 20.98 34 19797.2 2633.699 4.3883 10.45 35 19819.7 2628.754 4.3801 17.64 36 19850.7 2621.968 4.3688 26.32 37 19872.3 2617.228 4.3609 27.93 38 19897.2 2611.771 4.3518 17.82 39 19930.6 2604.438 4.3396 11.93 40 19963.7 2597.191 4.3275 8.30 41 20006.6 2587.791 4.3118 14.07 42 20031.7 2582.289 4.3027 11.75 43 20048.8 2578.545 4.2964 8.57 44 20076.5 2572.467 4.2863 11.71 45 20107.9 2565.584 4.2748 7.33 46 20151.6 2556.001 4.2589 14.11 47 20174.3 2551.024 4.2506 6.12 48 20196.3 2546.196 4.2425 13.56 49 20214.9 2542.128 4.2358 16.39 50 20247.5 2534.968 4.2238 26.96 51 20285.9 2526.555 4.2098 39.00 52 20305.4 2522.271 4.2027 24.70 53 20319.5 2519.190 4.1975 23.22 54 20346.4 2513.298 4.1877 7.91 55 20358.9 2510.546 4.1831 7.69 56 20379.8 2505.959 4.1755 8.17 57 20422.0 2496.719 4.1601 10.22 58 20461.6 2488.043 4.1456 5.87 59 20487.5 2482.352 4.1362 7.27 60 20514.7 2476.402 4.1262 7.42 61 20558.8 2466.727 4.1101 7.60 62 20596.2 2458.528 4.0965 31.95 63 20697.7 2436.266 4.0594 8.35 64 20735.2 2428.056 4.0457 13.12 65 20773.1 2419.740 4.0318 13.77 66 20811.2 2411.385 4.0179 12.76 67 20848.0 2403.322 4.0045 7.29 68 20890.7 2393.971 3.9889 7.87 69 20932.7 2384.749 3.9735 10.17 70 20970.3 2376.499 3.9598 12.68 71 21017.7 2366.108 3.9425 6.44 72 21123.5 2342.921 3.9038 16.37 73 21559.5 2247.327 3.7445 11.72 74 21604.4 2237.496 3.7282 11.07 75 22810.2 1973.141 3.2877 6.60 76 22829.6 1968.890 3.2806 6.56 77 22863.1 1961.542 3.2684 6.70 78 22883.0 1957.180 3.2611 6.59 79 24645.4 1570.792 2.6173 4.82 80 24691.2 1560.756 2.6006 5.30 81 25359.9 1414.147 2.3563 5.53 82 25407.3 1403.761 2.3390 10.56 83 25464.1 1391.301 2.3182 6.50 84 25529.5 1376.964 2.2943 2.87 85 25976.1 1279.056 2.1312 4.43 86 26038.8 1265.317 2.1083 9.07 87 26101.1 1251.649 2.0855 4.68 88 26348.4 1197.433 1.9952 2.18 89 26423.1 1181.067 1.9679 6.86 90 26495.7 1165.147 1.9414 40.57 91 26523.1 1159.138 1.9314 35.58 92 26528.4 1157.984 1.9295 36.42 93 26565.0 1149.953 1.9161 14.14 94 26612.0 1139.641 1.8989 12.71 95 26673.0 1126.283 1.8766 9.66 96 26829.5 1091.957 1.8194 48.60 97 26857.0 1085.929 1.8094 113.02 98 27349.4 977.978 1.6295 11.86 99 27403.6 966.099 1.6097 12.07 100 27963.7 843.313 1.4051 22.40 101 28018.6 831.275 1.3851 8.46 102 28254.5 779.546 1.2989 11.85 103 28298.3 769.948 1.2829 12.55 104 28317.6 765.717 1.2759 13.04 105 28356.2 757.269 1.2618 86.45 106 28387.8 750.328 1.2502 81.24 107 28464.0 733.633 1.2224 88.26 108 28533.4 718.414 1.1970 73.80 109 28677.9 686.739 1.1443 3.89 110 28745.9 671.820 1.1194 3.59 111 28867.8 645.098 1.0749 74.04 112 28987.7 618.804 1.0311 4.13 113 29051.5 604.826 1.0078 8.69 114 29106.8 592.703 0.9876 4.79 115 29207.1 570.711 0.9509 114.09 116 29320.8 545.779 0.9094 3.45 117 29410.0 526.225 0.8768 1.79 118 29492.8 508.068 0.8466 1.41 119 29857.9 428.030 0.7132 9.46 120 29911.7 416.233 0.6935 9.30

TABLE 8.6 13C NMR of R1 peak list DU = C:/Bruker/XWIN-NMR, USER = guest, NAME = chan, EXPNO = F1 = 176.659 ppm, F2 = 6.843 ppm, MI = 0.06 cm, MAXI = 10000.00 cm, FREQUENCY # ADDRESS [Hz] [PPM] INTENSITY 1 5310.9 25331.195 167.8559 0.12 2 8227.3 22634.219 149.9845 1.57 3 8251.8 22611.535 149.8342 11.79 4 8281.1 22584.428 149.6546 12.50 5 8310.4 22557.355 149.4752 12.30 6 9381.7 21566.594 142.9100 0.11 7 10403.1 20622.062 136.6511 0.14 8 10577.3 20460.975 135.5836 1.08 9 10596.8 20442.969 135.4643 5.78 10 10623.6 20418.133 135.2998 6.16 11 10650.4 20393.346 135.1355 6.01 12 11696.9 19425.617 128.7229 0.14 13 12537.9 18647.846 123.5690 2.11 14 12557.4 18629.844 123.4497 10.62 15 12584.3 18604.896 123.2844 11.25 16 12611.3 18580.010 123.1195 10.98 17 13444.2 17809.705 118.0151 0.11 18 14249.2 17065.322 113.0825 0.15 19 15319.0 16075.968 106.5266 0.21 20 15492.3 15915.713 105.4647 0.24 21 15509.0 15900.229 105.3621 0.25 22 15823.2 15609.646 103.4365 0.18 23 16269.0 15197.378 100.7047 0.16 24 18158.1 13450.413 89.1285 0.12 25 19683.7 12039.604 79.7798 0.13 26 19797.3 11934.502 79.0834 0.14 27 19929.2 11812.533 78.2752 0.23 28 19945.5 11797.527 78.1757 0.42 29 20197.3 11564.589 76.6322 0.15 30 20211.9 11551.142 76.5431 0.16 31 20226.2 11537.928 76.4555 0.14 32 20335.9 11436.434 75.7830 0.10 33 20410.2 11367.753 75.3279 0.13 34 20473.6 11309.069 74.9390 0.38 35 20617.2 11176.295 74.0592 0.13 36 20755.1 11048.805 73.2144 0.15 37 20910.0 10905.522 72.2649 0.21 38 21019.3 10804.420 71.5950 0.15 39 21047.5 10778.391 71.4225 0.37 40 21283.2 10560.453 69.9783 0.12 41 21320.7 10525.739 69.7483 0.12 42 21375.2 10475.339 69.4143 0.14 43 21430.8 10423.882 69.0733 0.13 44 22496.2 9438.663 62.5448 0.19 45 22510.2 9425.676 62.4588 0.20 46 23646.3 8375.039 55.4968 0.13 47 24915.8 7201.028 47.7173 0.13 48 25152.5 6982.204 46.2672 0.11 49 25671.3 6502.366 43.0876 0.12 50 25868.3 6320.202 41.8805 0.10 51 25921.2 6271.273 41.5563 0.11 52 26160.1 6050.418 40.0928 0.10 53 26300.3 5920.724 39.2334 0.13 54 26396.6 5831.667 38.6433 0.10 55 26722.2 5530.549 36.6479 0.11 56 26820.5 5439.679 36.0458 0.13 57 27398.1 4905.475 32.5059 0.10 58 27774.8 4557.162 30.1978 0.23 59 28148.7 4211.391 27.9066 0.23 60 28389.5 3988.645 26.4306 0.10 61 28448.2 3934.385 26.0710 0.17 62 28512.9 3874.571 25.6747 0.10 63 28828.2 3583.030 23.7428 0.11 64 29348.8 3101.522 20.5521 0.23 65 29529.3 2934.591 19.4459 0.22 66 29650.2 2822.798 18.7051 0.26 67 29719.5 2758.712 18.2805 0.11 68 29766.2 2715.508 17.9942 0.10 69 29970.4 2526.664 16.7428 0.24 70 29985.5 2512.765 16.6507 0.17 71 30140.7 2369.192 15.6993 0.22

TABLE 9.2 2D NMR (HMQC) chemical shift list of O54. Assignment w1 w2 Data Height 1 123.540 5.337 10801059 2 106.679 4.875 7546160 3 106.701 4.860 8527622 4 105.189 5.129 11004760 5 105.382 4.997 11249292 6 105.173 5.114 7915383 7 103.375 4.725 9431190 8 100.663 6.513 18941426 9 88.873 3.329 5975702 10 88.850 3.307 5246604 11 79.767 4.210 7199634 12 79.708 4.184 2885672 13 78.203 4.213 23826162 14 78.196 3.907 13786228 15 76.818 4.148 6859174 16 76.490 3.944 5773632 17 76.642 3.749 7515808 18 76.628 3.730 8437212 19 75.842 4.089 22251034 20 75.318 4.241 2654032 21 75.330 4.213 9853961 22 75.103 4.299 7580958 23 75.169 4.279 6376272 24 74.986 4.025 11823145 25 75.168 4.003 10800605 26 74.084 4.330 4901724 27 74.098 4.309 7830222 28 74.228 4.291 3975150 29 72.281 4.711 17310028 30 72.230 4.625 8709239 31 72.206 4.606 7361591 32 71.479 4.220 14407576 33 71.481 4.208 15081282 34 71.581 4.169 4582404 35 71.613 4.150 8419769 36 71.628 4.121 5804582 37 71.637 4.099 7950110 38 71.667 4.080 3119152 39 70.305 4.890 7622650 40 70.298 4.868 7628303 41 70.265 4.314 4762902 42 69.983 4.676 6227999 43 69.918 4.649 6037791 44 70.105 4.341 9963877 45 69.129 4.783 4704838 46 62.554 4.502 14998714 47 62.532 4.477 14154374 48 62.548 4.362 11342701 49 62.537 4.344 8091356 50 56.159 3.697 1838600 51 56.081 3.758 2144782 52 55.778 3.686 1783080 53 55.590 0.717 5572202 54 55.588 0.694 6671325 55 47.732 1.608 4217994 56 47.785 1.595 3965686 57 46.238 2.131 1734693 58 46.271 2.105 4919338 59 46.299 2.072 2490009 60 46.279 1.304 4826865 61 46.276 1.272 4573323 62 41.612 2.605 4436446 63 41.597 2.572 4591152 64 38.742 1.643 4256302 65 38.730 1.612 3985649 66 38.641 1.031 3538408 67 32.508 1.413 10863677 68 30.189 1.220 58134804 69 27.984 1.242 55524444 70 26.495 2.420 3113032 71 26.468 2.392 3183732 72 25.878 1.917 3435953 73 25.880 1.880 1979946 74 26.066 1.242 63345236 75 25.715 1.212 5054462 76 23.814 1.988 2104036 77 23.705 1.959 3237644 78 23.659 1.900 2754050 79 23.695 1.865 3108091 80 19.416 1.257 40949520 81 18.692 1.802 30867330 82 18.344 1.389 3540974 83 18.308 1.413 6204910 84 17.911 2.330 3260134 85 17.942 2.308 4212457 86 17.914 1.936 2468661 87 17.943 1.904 3914254 88 16.825 0.980 48392540 89 16.640 1.072 52924908 90 15.701 0.947 60375744

TABLE 9.3 2D NMR (HMBC) chemical shift list of O54. DU = C:/Bruker/XWIN-NMR, USER = guest, NAME = chan, EXPNO = 37, PROCNO = 1 F1PLO = 149.426 ppm, F1PHI = 11.307 ppm, F2PLO = 6.861 ppm, F2PHI = 0.388 ppm MI = 2.00 cm, MAXI = 10000.00 cm, PC = 3.000 ADDRESS FREQUENCY row [Hz]F1 [PPM]F1 # col [Hz]F2 [PPM]F2 INTENSITY 1 294 21551.752 142.8145 3.15 728 1553.288 2.5881 2 294 21551.752 142.8145 3.03 791 1185.345 1.9750 3 294 21551.752 142.8145 3.00 801 1127.853 1.8793 4 294 21551.752 142.8145 41.49 866 744.836 1.2411 5 392 18639.387 123.5154 2.70 728 1554.313 2.5898 6 392 18639.387 123.5154 2.11 730 1540.225 2.5664 7 392 18639.387 123.5154 2.94 794 1164.901 1.9410 8 392 18639.387 123.5154 3.61 801 1127.069 1.8779 9 478 16106.854 106.7334 20.41 585 2391.723 3.9851 10 478 16106.854 106.7334 3.17 653 1992.507 3.3200 11 485 15890.850 105.3020 3.34 494 2924.288 4.8725 12 485 15890.850 105.3020 3.27 516 2797.792 4.6617 13 485 15890.850 105.3020 5.99 547 2614.903 4.3570 14 485 15890.850 105.3020 11.83 583 2405.277 4.0077 15 495 15600.868 103.3804 4.55 560 2539.046 4.2306 16 495 15600.868 103.3804 3.63 574 2456.564 4.0932 17 509 15198.148 100.7118 11.15 312 3996.510 6.6591 18 509 15198.148 100.7118 12.11 341 3824.267 6.3721 19 509 15198.148 100.7118 2.82 560 2539.421 4.2312 20 570 13411.609 88.8731 18.81 495 2921.818 4.8684 21 570 13411.609 88.8731 2.73 797 1147.142 1.9114 22 570 13411.609 88.8731 6.25 827 975.260 1.6250 23 570 13411.609 88.8731 69.46 866 746.125 1.2432 24 570 13411.609 88.8731 48.36 892 589.846 0.9828 25 616 12031.100 79.7251 10.23 561 2533.072 4.2207 26 616 12031.100 79.7251 12.78 568 2492.076 4.1524 27 624 11792.174 78.1418 3.71 469 3075.630 5.1247 28 624 11792.174 78.1418 2.57 481 3002.844 5.0034 29 624 11792.174 78.1418 2.54 534 2690.732 4.4834 30 624 11792.174 78.1418 2.56 547 2617.207 4.3608 31 624 11792.174 78.1418 29.07 561 2532.765 4.2201 32 624 11792.174 78.1418 15.44 573 2460.286 4.0994 33 624 11792.174 78.1418 13.41 580 2419.846 4.0320 34 624 11792.174 78.1418 8.30 585 2390.343 3.9828 35 624 11792.174 78.1418 3.23 868 732.442 1.2204 36 632 11558.919 76.5961 11.58 553 2577.697 4.2950 37 632 11558.919 76.5961 3.02 563 2521.889 4.2020 38 632 11558.919 76.5961 13.81 573 2462.246 4.1026 39 632 11558.919 76.5961 9.11 861 772.953 1.2879 40 632 11558.919 76.5961 33.06 864 755.650 1.2591 41 632 11558.919 76.5961 53.66 868 732.170 1.2200 42 639 11373.424 75.3669 5.00 326 3911.898 6.5181 43 639 11373.424 75.3669 25.40 563 2523.630 4.2049 44 639 11373.424 75.3669 5.16 574 2456.045 4.0923 45 639 11373.424 75.3669 5.57 611 2240.865 3.7338 46 639 11373.424 75.3669 2.40 808 1082.299 1.8034 47 639 11373.424 75.3669 2.78 864 756.608 1.2607 48 639 11373.424 75.3669 5.47 868 732.370 1.2203 49 646 11168.377 74.0082 8.44 511 2828.344 4.7127 50 646 11168.377 74.0082 6.66 520 2772.974 4.6204 51 646 11168.377 74.0082 5.35 563 2524.284 4.2060 52 646 11168.377 74.0082 49.69 808 1082.395 1.8035 53 655 10899.106 72.2238 25.27 326 3911.679 6.5177 54 655 10899.106 72.2238 2.42 498 2902.662 4.8365 55 655 10899.106 72.2238 5.76 511 2829.414 4.7144 56 655 10899.106 72.2238 2.33 523 2756.376 4.5927 57 655 10899.106 72.2238 2.40 532 2703.575 4.5048 58 655 10899.106 72.2238 9.99 552 2588.313 4.3127 59 655 10899.106 72.2238 7.35 563 2523.240 4.2043 60 655 10899.106 72.2238 2.26 808 1083.933 1.8061 61 659 10785.637 71.4719 2.28 326 3912.133 6.5185 62 659 10785.637 71.4719 2.36 494 2925.999 4.8754 63 659 10785.637 71.4719 3.39 516 2800.177 4.6657 64 659 10785.637 71.4719 7.49 532 2702.493 4.5030 65 659 10785.637 71.4719 7.86 534 2691.698 4.4850 66 659 10785.637 71.4719 2.35 547 2614.980 4.3571 67 659 10785.637 71.4719 46.85 562 2525.201 4.2075 68 659 10785.637 71.4719 2.02 584 2398.095 3.9958 69 665 10604.599 70.2723 2.12 326 3911.956 6.5182 70 665 10604.599 70.2723 18.68 469 3075.252 5.1241 71 665 10604.599 70.2723 3.29 481 3002.048 5.0021 72 665 10604.599 70.2723 3.03 562 2525.138 4.2074 73 665 10604.599 70.2723 6.46 573 2460.794 4.1002 74 665 10604.599 70.2723 3.10 808 1081.934 1.8027 75 666 10552.990 69.9303 2.10 326 3912.459 6.5190 76 666 10552.990 69.9303 13.23 468 3077.187 5.1273 77 666 10552.990 69.9303 10.72 481 3002.402 5.0027 78 666 10552.990 69.9303 3.33 563 2522.129 4.2024 79 666 10552.990 69.9303 4.47 573 2461.269 4.1010 80 666 10552.990 69.9303 2.72 808 1081.723 1.8024 81 671 10429.136 69.1095 38.49 326 3911.639 6.5177 82 671 10429.136 69.1095 8.97 552 2589.015 4.3139 83 671 10429.136 69.1095 63.77 808 1081.987 1.8028 84 705 9425.537 62.4591 3.27 560 2542.206 4.2359 85 705 9425.537 62.4591 2.08 562 2526.304 4.2094 86 740 8376.884 55.5101 3.45 827 971.239 1.6183 87 740 8376.884 55.5101 3.43 849 843.561 1.4056 88 740 8376.884 55.5101 48.00 866 746.292 1.2435 89 740 8376.884 55.5101 31.95 892 590.131 0.9833 90 740 8376.884 55.5101 25.60 896 567.050 0.9448 91 780 7196.908 47.6909 4.85 447 3202.461 5.3360 92 780 7196.908 47.6909 2.83 864 756.127 1.2599 93 780 7196.908 47.6909 3.78 868 731.658 1.2191 94 780 7196.908 47.6909 28.25 883 642.229 1.0701 95 780 7196.908 47.6909 32.38 896 567.166 0.9450 96 780 7196.908 47.6909 2.37 922 414.895 0.6913 97 788 6979.996 46.2535 2.37 730 1540.055 2.5661 98 788 6979.996 46.2535 58.65 864 755.679 1.2591 99 788 6979.996 46.2535 85.12 868 731.892 1.2195 100 804 6505.572 43.1097 4.16 553 2577.654 4.2949 101 804 6505.572 43.1097 4.70 574 2455.343 4.0911 102 804 6505.572 43.1097 3.13 755 1393.963 2.3227 103 804 6505.572 43.1097 3.35 858 789.603 1.3157 104 804 6505.572 43.1097 9.90 861 773.435 1.2887 105 804 6505.572 43.1097 9.41 867 737.064 1.2281 106 804 6505.572 43.1097 2.74 883 642.755 1.0710 107 804 6505.572 43.1097 2.09 892 589.037 0.9815 108 810 6311.923 41.8265 8.24 447 3202.295 5.3357 109 810 6311.923 41.8265 6.43 755 1393.660 2.3221 110 810 6311.923 41.8265 2.54 830 955.929 1.5928 111 810 6311.923 41.8265 45.72 866 744.369 1.2403 112 810 6311.923 41.8265 3.80 869 724.741 1.2076 113 810 6311.923 41.8265 53.84 883 642.271 1.0702 114 810 6311.923 41.8265 2.36 892 589.734 0.9826 115 814 6190.622 41.0227 2.27 447 3204.169 5.3389 116 814 6190.622 41.0227 4.20 866 744.967 1.2413 117 814 6190.622 41.0227 13.51 883 641.291 1.0685 118 814 6190.622 41.0227 3.54 892 590.266 0.9835 119 817 6122.212 40.5694 2.02 849 842.541 1.4039 120 817 6122.212 40.5694 10.92 866 744.098 1.2398 121 817 6122.212 40.5694 19.65 883 641.844 1.0695 122 817 6122.212 40.5694 4.84 892 590.010 0.9831 123 819 6054.448 40.1203 2.01 800 1128.653 1.8806 124 819 6054.448 40.1203 5.24 830 955.874 1.5927 125 819 6054.448 40.1203 7.20 849 842.438 1.4037 126 819 6054.448 40.1203 39.00 866 744.338 1.2402 127 819 6054.448 40.1203 49.93 883 642.047 1.0698 128 819 6054.448 40.1203 6.64 892 589.724 0.9826 129 819 6054.448 40.1203 2.44 896 566.654 0.9442 130 823 5927.002 39.2758 48.54 866 746.066 1.2431 131 823 5927.002 39.2758 7.22 883 642.112 1.0699 132 823 5927.002 39.2758 60.87 892 589.816 0.9828 133 823 5927.002 39.2758 5.86 896 566.631 0.9441 134 823 5927.002 39.2758 7.24 922 415.473 0.6923 135 827 5833.933 38.6590 3.11 827 970.335 1.6168 136 827 5833.933 38.6590 10.44 865 746.825 1.2444 137 827 5833.933 38.6590 3.42 883 642.035 1.0698 138 827 5833.933 38.6590 11.20 892 588.957 0.9813 139 827 5833.933 38.6590 55.77 896 567.124 0.9450 140 830 5737.570 38.0205 3.79 864 753.968 1.2563 141 830 5737.570 38.0205 4.47 868 731.483 1.2188 142 830 5737.570 38.0205 2.21 883 641.875 1.0695 143 830 5737.570 38.0205 4.70 892 589.272 0.9819 144 830 5737.570 38.0205 11.09 896 567.206 0.9451 145 833 5652.819 37.4589 5.17 865 751.942 1.2529 146 833 5652.819 37.4589 5.78 868 732.193 1.2200 147 833 5652.819 37.4589 2.64 883 642.313 1.0702 148 833 5652.819 37.4589 2.92 892 590.189 0.9834 149 833 5652.819 37.4589 11.22 896 567.196 0.9451 150 837 5532.757 36.6633 2.33 747 1441.047 2.4011 151 837 5532.757 36.6633 6.70 830 956.266 1.5934 152 837 5532.757 36.6633 7.18 849 840.791 1.4009 153 837 5532.757 36.6633 10.60 864 754.417 1.2570 154 837 5532.757 36.6633 10.09 868 733.768 1.2226 155 837 5532.757 36.6633 2.62 883 642.220 1.0701 156 837 5532.757 36.6633 59.49 896 567.041 0.9448 157 837 5532.757 36.6633 7.27 922 415.510 0.6923 158 840 5447.502 36.0983 3.44 611 2241.469 3.7348 159 840 5447.502 36.0983 2.18 775 1277.736 2.1290 160 840 5447.502 36.0983 5.77 778 1261.898 2.1026 161 840 5447.502 36.0983 2.77 780 1246.496 2.0769 162 840 5447.502 36.0983 7.30 861 771.393 1.2853 163 840 5447.502 36.0983 85.50 864 755.513 1.2589 164 840 5447.502 36.0983 94.68 868 731.684 1.2191 165 840 5447.502 36.0983 2.09 883 642.211 1.0701 166 840 5447.502 36.0983 11.25 896 566.770 0.9444 167 858 4901.029 32.4771 2.31 859 783.807 1.3060 168 858 4901.029 32.4771 38.68 883 642.014 1.0697 169 858 4901.029 32.4771 3.35 922 416.874 0.6946 170 870 4554.428 30.1803 9.79 611 2241.599 3.7350 171 870 4554.428 30.1803 3.21 777 1263.228 2.1048 172 870 4554.428 30.1803 18.60 857 793.884 1.3228 173 870 4554.428 30.1803 93.90 864 755.200 1.2583 174 870 4554.428 30.1803 19.16 879 668.641 1.1141 175 870 4554.428 30.1803 3.11 892 590.200 0.9834 176 881 4218.921 27.9570 16.41 855 807.262 1.3451 177 881 4218.921 27.9570 4.75 864 755.803 1.2593 178 881 4218.921 27.9570 15.67 876 683.031 1.1381 179 881 4218.921 27.9570 73.26 892 589.918 0.9829 180 881 4218.921 27.9570 2.87 922 416.446 0.6939 181 891 3935.921 26.0817 2.34 802 1120.176 1.8665 182 891 3935.921 26.0817 19.11 855 806.480 1.3438 183 891 3935.921 26.0817 8.92 866 745.923 1.2429 184 891 3935.921 26.0817 5.17 868 731.989 1.2197 185 891 3935.921 26.0817 18.56 877 680.851 1.1344 186 891 3935.921 26.0817 4.65 892 589.687 0.9825 187 893 3878.913 25.7039 2.05 791 1185.608 1.9755 188 893 3878.913 25.7039 2.02 810 1072.605 1.7872 189 893 3878.913 25.7039 4.93 855 805.970 1.3429 190 893 3878.913 25.7039 44.75 866 744.248 1.2401 191 893 3878.913 25.7039 4.45 877 680.894 1.1345 192 893 3878.913 25.7039 3.96 892 590.170 0.9834 193 925 2932.829 19.4346 15.68 611 2240.954 3.7339 194 925 2932.829 19.4346 4.57 775 1278.753 2.1307 195 925 2932.829 19.4346 11.84 778 1262.372 2.1034 196 925 2932.829 19.4346 6.85 780 1246.392 2.0768 197 925 2932.829 19.4346 22.66 854 816.734 1.3609 198 925 2932.829 19.4346 93.22 868 732.145 1.2199 199 925 2932.829 19.4346 21.54 875 691.416 1.1521 200 933 2709.260 17.9531 10.65 553 2577.918 4.2954 201 933 2709.260 17.9531 2.40 574 2456.969 4.0939 202 933 2709.260 17.9531 2.47 728 1553.812 2.5890 203 933 2709.260 17.9531 2.44 786 1214.060 2.0229 204 933 2709.260 17.9531 4.64 865 746.759 1.2443 205 933 2709.260 17.9531 5.50 868 731.918 1.2195 206 938 2536.520 16.8085 2.61 653 1992.054 3.3192 207 938 2536.520 16.8085 2.70 829 958.476 1.5970 208 938 2536.520 16.8085 52.11 866 745.904 1.2428 209 938 2536.520 16.8085 17.18 882 650.673 1.0842 210 938 2536.520 16.8085 3.28 895 574.736 0.9576 211 938 2536.520 16.8085 16.84 903 526.041 0.8765 212 938 2536.520 16.8085 8.32 922 416.280 0.6936 213 944 2375.487 15.7414 5.34 830 955.713 1.5924 214 944 2375.487 15.7414 6.94 866 745.684 1.2425 215 944 2375.487 15.7414 21.31 886 627.576 1.0457 216 944 2375.487 15.7414 3.00 892 588.774 0.9810 217 944 2375.487 15.7414 21.33 907 503.819 0.8395 218 944 2375.487 15.7414 10.43 922 415.374 0.6921

TABLE 9.5 Proton NMR peak list of O54. DU = C:/Bruker/XWIN-NMR, USER = guest, NAME = chan, EXPNO = 35, PROCNO = 1 F1 = 10.000 ppm, F2 = 0.000 ppm, MI = 2.00 cm, MAXI = 10000.00 cm, PC = 1.000 FREQUENCY # ADDRESS [Hz] [PPM] INTENSITY 1 7886.3 5225.509 8.7069 180.00 2 11038.4 4534.438 7.5554 72.73 3 12044.3 4313.911 7.1879 160.90 4 13878.4 3911.819 6.5180 14.53 5 17112.8 3202.719 5.3364 9.02 6 17674.0 3079.680 5.1314 10.52 7 17709.4 3071.938 5.1185 10.66 8 18004.6 3007.215 5.0107 10.57 9 18039.9 2999.480 4.9978 10.78 10 18330.8 2935.701 4.8915 6.88 11 18377.3 2925.509 4.8745 16.06 12 18411.1 2918.099 4.8622 10.01 13 18591.4 2878.554 4.7963 3.55 14 18621.6 2871.941 4.7853 4.53 15 18660.9 2863.325 4.7709 3.56 16 18768.9 2839.657 4.7315 8.48 17 18801.9 2832.416 4.7194 15.95 18 18929.2 2804.496 4.6729 5.72 19 18974.3 2794.608 4.6564 6.90 20 19049.2 2778.191 4.6291 5.41 21 19063.7 2775.007 4.6238 4.83 22 19091.3 2768.955 4.6137 5.79 23 19106.0 2765.737 4.6083 5.24 24 19395.7 2702.228 4.5025 11.91 25 19447.9 2690.790 4.4835 16.03 26 19708.9 2633.561 4.3881 6.86 27 19731.8 2628.552 4.3798 8.37 28 19761.7 2621.989 4.3688 16.78 29 19784.1 2617.071 4.3606 18.07 30 19814.8 2610.350 4.3494 12.73 31 19834.7 2605.976 4.3421 13.55 32 19863.8 2599.612 4.3315 9.52 33 19909.8 2589.523 4.3147 13.96 34 19947.5 2581.249 4.3009 10.27 35 19990.6 2571.809 4.2852 7.86 36 20116.2 2544.275 4.2393 6.06 37 20155.1 2535.750 4.2251 29.53 38 20188.0 2528.527 4.2131 36.53 39 20218.4 2521.859 4.2020 22.79 40 20258.5 2513.072 4.1873 10.10 41 20308.5 2502.111 4.1691 7.17 42 20349.0 2493.228 4.1543 10.94 43 20384.7 2485.412 4.1412 7.98 44 20454.4 2470.136 4.1158 8.00 45 20496.1 2460.979 4.1005 11.54 46 20522.9 2455.108 4.0908 20.70 47 20619.4 2433.953 4.0555 5.10 48 20655.0 2426.151 4.0425 7.46 49 20704.6 2415.271 4.0244 6.32 50 20741.8 2407.119 4.0108 8.93 51 20763.7 2402.312 4.0028 6.78 52 20800.3 2394.290 3.9894 7.59 53 20840.0 2385.587 3.9749 4.23 54 20899.5 2372.534 3.9532 5.04 55 20961.5 2358.953 3.9305 5.93 56 21011.8 2347.917 3.9122 8.88 57 21462.4 2249.135 3.7476 8.28 58 21507.4 2239.268 3.7311 7.60 59 22606.3 1998.348 3.3297 4.57 60 22625.1 1994.242 3.3228 4.57 61 22658.9 1986.819 3.3105 4.74 62 22678.2 1982.598 3.3034 4.55 63 24608.6 1559.375 2.5983 3.63 64 24657.0 1548.768 2.5806 3.98 65 25113.6 1448.658 2.4138 3.70 66 25160.1 1438.461 2.3968 4.03 67 25341.7 1398.662 2.3305 3.88 68 25394.7 1387.036 2.3111 4.43 69 25900.2 1276.207 2.1264 3.15 70 25962.8 1262.484 2.1036 6.25 71 26025.4 1248.772 2.0807 3.23 72 26432.4 1159.530 1.9320 8.83 73 26518.7 1140.610 1.9005 11.87 74 26686.8 1103.771 1.8391 2.00 75 26772.7 1084.927 1.8077 28.83 76 26800.2 1078.904 1.7977 28.10 77 27239.8 982.535 1.6371 4.71 78 27287.4 972.099 1.6197 8.43 79 27366.6 954.736 1.5908 4.35 80 27864.2 845.631 1.4090 14.94 81 28172.8 777.969 1.2963 9.42 82 28216.7 768.363 1.2803 9.50 83 28235.2 764.288 1.2735 9.84 84 28276.7 755.206 1.2583 58.74 85 28321.2 745.447 1.2421 94.67 86 28380.5 732.444 1.2204 58.53 87 28793.1 641.978 1.0697 50.29 88 28906.6 617.114 1.0282 5.31 89 29031.6 589.696 0.9826 56.49 90 29134.1 567.228 0.9451 50.09 91 29765.3 428.852 0.7146 6.67 92 29818.8 417.128 0.6950 6.36

REFERENCES

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What is claimed is: 1-223. (canceled)
 224. A method for inhibiting cancer cell growth, comprising contacting the cells with an effective amount of saponins extract from Wenguanguo or Xanthoceras Sorbifolia, wherein the cancer is selected from the group consisting of breast cancer, leukocyte cancer, liver cancer, ovary cancer, bladder cancer, prostate cancer, bone cancer, colon cancer, cervix cancer, lung cancer and brain cancer, wherein the saponins effective drug concentration is 40-1 05 ug/ml for breast cancer, 25-50 ug/ml for leukocyte cancer, 35-55 ug/ml for bladder cancer, 10-20 ug/ml for ovarian cancer, 50-100 ug/ml for brain cancer, 20-70 ug/ml for prostate cancer, 90-1 30 ug/ml for lung cancer, 50-90 ug/ml for liver cancer, and 90-1 60 ug/ml for cervix cancer.
 225. The method of claim 224, wherein the cancer is breast cancer.
 226. The method of claim 224, wherein the cancer is leukocyte cancer.
 227. The method of claim 224, wherein the cancer is liver cancer.
 228. The method of claim 224, wherein the cancer is ovary cancer.
 229. The method of claim 224, wherein the cancer is prostate cancer.
 230. The method of claim 224, wherein the cancer is bone cancer.
 231. The method of claim 224, wherein the cancer is brain cancer.
 232. The method of claim 224, wherein the cancer is colon cancer.
 233. The method of claim 224, wherein the cancer is cervix cancer.
 234. The method of claim 224, wherein the cancer is lung cancer.
 235. The method of claim 224, wherein the saponins extracts are formulated with a pharmaceutically suitable carrier.
 236. The method of claim 224, wherein the saponins are selected from the profile of FIG.
 6. 